Methods and compositions for treating diseases and conditions

ABSTRACT

Provided herein include methods and compositions for treating diseases or conditions. In some embodiments provided are methods for treating one or more diseases or conditions selected from the group consisting of hypertension, heart failure, dyspnea, and sleep apnea. In certain embodiments provided are methods that include administering a compound of formula (I) as disclosed herein. In some embodiments provided are methods that include administering a P2X3 and/or a P2X2/3 receptor antagonist.

CROSS REFERENCE

This application claims priority to and the benefit of U.S. ProvisionalPatent Application Ser. No. 62/020,839, filed on Jul. 3, 2014, and U.S.Provisional Patent Application Ser. No. 62/120,643, filed on Feb. 25,2015, each of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to the field of altering physiologicalconditions and or treating diseases.

BACKGROUND

The information provided herein and references cited are provided solelyto assist the understanding of the reader, and does not constitute anadmission that any of the references or information is prior art to thepresent invention.

U.S. patent application Ser. No. 11/906,802 (Patented as U.S. Pat. No.8,003,788) discloses methods and intermediates for the preparation ofcompounds that are P2X3 and P2X2/3 receptor modulators. U.S. applicationSer. No. 11/187,753 discloses methods of diagnosing and treatingpulmonary diseases using certain specific P2 receptor modulators.

SUMMARY

The present disclosure relates to the field of altering physiologicalconditions and/or treating diseases.

In a first aspect, provided is a method of treating hypertension in asubject involving identifying a subject diagnosed with hypertension andadministering to the subject a compound formula (I), such as, forexample, one or more compounds selected from the group consisting of thecompounds of Table 1.

The term “hypertension” as used herein refers to a condition or diseasewell known in the art in which the blood pressure in a mammal ischronically elevated. In certain embodiments hypertension may refer to acondition in which a subject's resting systolic blood pressure is aboveabout 120 mmHg and/or diastolic pressure is above about 80 mmHg. Incertain embodiments hypertension may refer to a condition in which asubject's resting systolic blood pressure is above about 115 mmHg; orabove about 120 mmHg; or above about 125 mmHg; or above about 130 mmHg;or above about 135 mmHg; or above about 140 mmHg; or above about 145mmHg; or above about 150 mmHg; or above about 155; or above about 160;or above about 165; or above about 170 and/or resting diastolic pressureis above about 75 mmHg; or above about 80 mmHg; or above about 85 mmHg;or above about 90 mmHg; or above about 95 mmHg; or above about 100 mmHg;or above about 105 mmHg; or above about 110 mmHg. In some embodimentshypertension may be primary or secondary hypertension. In someembodiments hypertension may be chronic treatment resistanthypertension, defined as persistent hypertension (resting office bloodpressure>140/90 [SBP/DBP]) despite use of >3 antihypertensivemedications including a diuretic, as well as hypertension in patientsunable to tolerate currently preferred antihypertensive medications, orin whom approved medications cannot achieve recommended levels of BPcontrol. Diagnosis of hypertension in a subject may in variousembodiments be performed by an individual qualified to make suchdiagnosis in a particular jurisdiction.

In another aspect, provided is a method of treating heart failure in asubject involving identifying a subject diagnosed with heart failure andadministering to the subject a compound formula (I), such as, forexample, one or more compounds selected from the group consisting of thecompounds of Table 1.

The term “heart failure” as used herein refers to a condition or diseasewell known in the art which is associated with the heart being unable tomaintain blood flow sufficient to maintain the needs of the body.Diagnosis of heart failure may in certain embodiments be based onechocardiography results characteristic of heart failure. In someembodiments, heart failure may refer to a condition often referred to ascongestive heart failure. In some embodiments, heart failure may referto systolic heart failure, also called heart failure due to reducedejection fraction (HFREF) or heart failure due to left ventricularsystolic dysfunction. In some embodiments, heart failure may refer toheart failure with preserved ejection fraction (HFPEF) also known asdiastolic heart failure or heart failure with normal ejection fraction(HFNEF). In some embodiments, heart failure may be chronic heart failureand in other embodiments the heart failure may be acute heart failure.Diagnosis of heart failure in a subject may in various embodiments beperformed by an individual qualified to make such diagnosis in aparticular jurisdiction.

In a further aspect, provided is a method of treating dyspnea in asubject involving identifying a subject diagnosed with dyspnea andadministering to the subject a compound formula (I), such as, forexample, one or more compounds selected from the group consisting of thecompounds of Table 1.

The term “dyspnea” as used herein refers to a condition or disease wellknown in the art in which a subject experiences feelings or sensationsassociated with impaired breathing. In some embodiments dyspnea mayrefer to a condition consistent with the America Thoracic Societydefinition of dyspnea, i.e., “a subjective experience of breathingdiscomfort that consists of qualitatively distinct sensations that varyin intensity”. In some embodiments dyspnea may refer to sensations ofinadequate breathing, uncomfortable awareness of breathing and/orbreathlessness. Diagnosis of dyspnea in a subject may in variousembodiments be performed by an individual qualified to make suchdiagnosis in a particular jurisdiction.

In another aspect, provided is a method of treating sleep apnea in asubject involving identifying a subject diagnosed with sleep apnea andadministering to the subject a compound formula (I), such as, forexample, one or more compounds selected from the group consisting of thecompounds of Table 1.

The term “sleep apnea” as used herein refers to a condition or diseasewell known in the art characterized by disruptions in breathing (e.g.,pauses in breathing or instances of shallow or infrequent breathing)during sleep. In some aspects sleep apnea is central sleep apnea,obstructive sleep apnea, or mixed sleep apnea. In some embodiments,sleep apnea may be characterized by more than about 5 apneic events perhour of sleep; or more than about 10 apneic events per hour of sleep; ormore than about 15 apneic events per hour sleep; or more than about 20apneic events per hour of sleep, or more than about 25 apneic events perhour of sleep, or more than about 30 apneic sleep events per hour sleep;or more than about 35 apneic sleep events per hour sleep. Diagnosis ofdyspnea in a subject may in various embodiments be performed by anindividual qualified to make such diagnosis in a particularjurisdiction.

In one aspect, provided is a method for altering carotid body tonicityor activity in a subject, involving identifying a subject in need ofaltering carotid body tonicity or activity and administering to thesubject a compound formula (I), such as, for example, one or morecompounds selected from the group consisting of the compounds of Table1.

The term “carotid body” as used herein refers to a small cluster ofchemoreceptors and supporting cells located near the fork (bifurcation)of the carotid artery. The carotid body is also referred in the art ascarotid glomus or glomus caroticum. The term “altering carotid bodytonicity” or activity as used herein means modifying the level ofexcitation of carotid sinus nerve chemoreceptor afferents that aredischarging excessively in response to dysregulated levels of arterialchemicals (hyperreflexia), as well as attenuating the aberrant,spontaneous discharge of such nerve fibers that can occur in the absenceof chemical dysregulation.

In one aspect, provided is a method for reducing carotid body tonicityin a subject involving identifying a subject in need of reduction incarotid body tonicity or activity and administering to the subject acompound of formula (I), such as, for example, one or more compoundsselected from the group consisting of the compounds of Table 1.

In an additional aspect, provided is a method of reducing carotid bodychemosensory afferent discharge in a subject involving identifying asubject in need of reduction in carotid body chemosensory afferentdischarge and administering to the subject a compound formula (I), suchas, for example, one or more compounds selected from the groupconsisting of the compounds of Table 1.

The term “carotid body chemosensory afferent discharge” as used hereinmeans the electrical generation of action potentials from peripheralcarotid sinus nerve terminals abutting glomus cells in the carotid body,and their orthograde propagation to petrosal ganglion perikarya as wellas afferent central terminal projections located within the medullarynucleus tractus solitarius (NTS) in the brainstem.

The term “a compound of formula (I) as used herein refers to compoundshaving a structure of the below formula (I):

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹ is hydrogen or optionally substituted alkyl, andR² is alkyl; alkenyl; alkynyl; amino; aminosulfonyl; halo; amido;haloalkyl; alkoxy; hydroxy; haloalkoxy; nitro; hydroxyalkyl;alkoxyalkyl; hydroxyalkoxy; alkynylalkoxy; alkylsulfonyl; arylsulfonyl;carboxyalkyl; cyano or alkylcarbonyl. In one or more embodiments of anyaspects of the invention, R¹ can be methyl or R¹ can be hydrogen. In oneor more embodiments R² can be haloalkyl, aminosulfonyl, alkylsulfonyl,alkylcarbonyl or carboxyalkyl. In some embodiments, R² is haloalkyl,wherein the alkyl is methyl.

In some embodiments, R² is aminosulfonyl. R² is carboxyalkyl or R² isalkylcarbonyl. In certain embodiments of formula (I), R¹ is methyl. Incertain embodiments of formula (I), R¹ is hydrogen. In certainembodiments of formula (I), R² is haloalkyl, aminosulfonyl,alkylsulfonyl, alkylcarbonyl or carboxyalkyl. In certain embodiments offormula (I), R² is haloalkyl, where alkyl is methyl. In certainembodiments of formula (I), R² is aminosulfonyl. In certain embodimentsof formula (I), R² is carboxyalkyl. In certain embodiments of formula(I), R² is alkylcarbonyl. Where R¹, or R², is alkyl or contains an alkylmoiety, such alkyl is preferably lower alkyl, i.e. C₁-C₆alkyl, and morepreferably C₁-C₄alkyl. Compounds of formula I may be synthesized forexample as disclosed in U.S. Pat. No. 8,808,313. In some embodiments, acompound of formula (I) is a compound selected from Table I.

The term “subject” as used herein refers to a biological organism. Incertain embodiments a subject may be a mammal or non-mammal animal.Mammals means any member of the mammalia class including, but notlimited to, humans; non-human primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, horses, sheep,goats, and swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice, and guineapigs; and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. The term “subject” does not denote aparticular age or sex. In certain embodiments the individual may be apatient, i.e., an subject that is a candidate for, or awaiting, medicalor other treatment, such as, e.g., the methods as described herein. Apatient may, in some embodiments, be a human patient or a veterinarypatient.

The terms “treating,” “treat,” “treatment,” “treatable” and the likerefer to: modulating (e.g., preventing or reducing) a disease, disorderor condition from occurring in a cell, a tissue, a system, animal orsubject; stabilizing a disease, disorder or condition, i.e., arrestingits development; and/or modulating (e.g., reducing or alleviating) oneor more symptoms of the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition. It is understoodthat the term “treating” as contemplated herein is used in the contextof a statistical sample or an average subject and in some embodimentsmay not require an observable effect in all subjects or samplessubjected to a particular therapeutic, agent (such as a P2X3 or P2X2/3modulator) or method. The term treating or treatment as used herein mayin certain embodiments involve a step of identifying a subject ashaving, being suspected of having and/or being diagnosed with a diseaseor condition to be treated. As used herein, a therapeutic that“prevents” a disorder or condition refers to a compound that, in astatistical sample, reduces the occurrence of the disorder or conditionin the treated sample relative to an untreated control sample, or delaysthe onset or reduces the severity of one or more symptoms of thedisorder or condition relative to the untreated control sample. It isunderstood that the term “prevents” as contemplated herein is used inthe context of a statistical sample or an average subject and in someembodiments may not require an observable effect in all subjects orsamples subjected to a particular therapeutic or agent.

“Therapeutically effective amount” or “effective amount” means an amountof a compound or agent (such as, e.g., a P2X3 and/or P2X2/3 modulator)that, when administered to a subject for a particular purpose, issufficient to modulate such intended purpose. The “therapeuticallyeffective amount” will vary depending on the compound, disease statebeing treated, the severity or the disease treated, the age and relativehealth of the subject, the route and form of administration, thejudgment of the attending medical or veterinary practitioner, and otherfactors. It is understood that the term “therapeutically effectiveamount” as contemplated herein is used in the context of a statisticalsample or an average subject and in some embodiments may not require anobservable effect in all subjects or samples subjected to a particularcompound or agent. In various aspects and embodiments contemplatedherein, administering a compound refers to administering atherapeutically effective amount of such compound.

The term “about” as used herein means in quantitative terms plus orminus 10%. For example, “about 3%” would encompass 2.7-3.3% and “about10%” would encompass 9-11%. In some embodiments the term about means inquantitative terms plus or minus 8%; or 5% or 3% or 1%. Moreover, where“about” is used herein in conjunction with a quantitative term it isunderstood that in addition to the value plus or minus 10%, the exactvalue of the quantitative term is also contemplated and described. Forexample, the term “about 3%” expressly contemplates, describes andincludes exactly 3%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows a proof-of-concept experiment involving compoundA. FIG. 1A shows the effects of Compound A, on carotid sinus nerveactivity and excitatory responses to hypoxia and ATP (or analogs), in awild-type mouse. FIG. 1B shows the effects of Compound A on carotidsinus nerve activity and excitatory responses to hypoxia and ATP (oranalogs) in a P2X2-gene deletion mutant (P2X2KO) mouse. FIG. 1C showsthe effects of Compound A on carotid sinus nerve activity and excitatoryresponses to hypoxia and ATP (or analogs) in a P2X3-gene deletion mutant(P2X3KO) mouse.

FIG. 2A shows denervation of the carotid body chemosensors in consciousSHRs (compared with normotensive Wistar rats) markedly reduces arterialblood pressure and renal sympathetic nerve activity.

FIG. 2B SHRs show characteristic tonic activity in petrosal ganglionneuronal cell bodies of carotid sinus afferents, as well as exaggeratedresponses to chemical provocation with NaCN, that are absent or lessenedin Wistar rats.

FIG. 2C shows that significant reductions in arterial blood pressure inSHRs are seen following bolus dosing of a Compound of formula I (1mg/kg), which are absent in SHRs following carotid body ablation as wellas in normotensive Wistar rats.

FIG. 2D illustrates immunocytochemical labelling of a section of carotidbody from a hypertensive human subject.

DETAILED DESCRIPTION

Certain aspects, embodiments and/or methods described herein are basedat least in part on the surprising finding that compounds of formula (I)have surprising affects on certain diseases and conditions.

Non-limiting exemplary compounds in accordance with the methods,technologies and disclosures provided herein are shown in Table 1.

TABLE 1 # Structure Name 1

5-(2-Isopropyl-4,5-dimethoxy- phenoxy)-pyrimidine-2,4-diamine 2

5-(5-Bromo-2-isopropyl-4-methoxy- phenoxy)-pyrimidine-2,4-diamine 3

5-(5-Chloro-2-isopropyl-4-methoxy- phenoxy)-pyrimidine-2,4-diamine 4

5-(2-Isopropyl-4-methoxy-5-methyl- phenoxy)-pyrimidine-2,4-diamine 5

1-[5-(2,4-Diamino-pyrimidin-5- yloxy)-4-isopropyl-2-methoxy-phenyl]-ethanone 6

5-(2,4-Diamino-pyrimidin-5-yloxy)- 4-isopropyl-2-methoxy-benzamide 7

5-(2,4-Diamino-pyrimidin-5-yloxy)- 4-isopropyl-2-methoxy-benzoic acid 8

5-(2,4-Diamino-pyrimidin-5-yloxy)- 4-isopropyl-2-methoxy-benzonitrile 9

[5-(2,4-Diamino-pyrimidin-5-yloxy)- 4-isopropyl-2-methoxy-phenyl]-urea10

5-(5-Chloro-4-difluoromethoxy-2- isopropyl-phenoxy)-pyrimidine-2,4-diamine 11

5-(5-Amino-2-isopropyl-4-methoxy- phenoxy)-pyrimidine-2,4-diamine 12

N-[5-(2,4-Diamino-pyrimidin-5- yloxy)-4-isopropyl-2-methoxy-phenyl]-acetamide 13

5-(2-Isopropyl-5-methanesulfonyl-4- methoxy-phenoxy)-pyrimidine-2,4-diamine 14

1-[5-(2,4-Diamino-pyrimidin-5- yloxy)-2-hydroxy-4-isopropyl-phenyl]-ethanone 15

5-(5-Iodo-2-isopropyl-4-methoxy- phenoxy)-pyrimidine-2,4-diamine 16

5-(2,4-Diamino-pyrimidin-5-yloxy)- 4-isopropyl-2-methoxy-benzenesulfonamide 17

4-(2,4-Diamino-pyrimidin-5-yloxy)- 2-iodo-5-isopropyl-phenol 18

5-(2-Isopropyl-4methoxy-5-vinyl- phenoxy)-pyrimidine-2,4-diamine 19

5-(2-Isopropyl-4-methoxy-5- trifluoromethyl-phenoxy)-pyrimidine-2,4-diamine 20

1-[5-(2,4-Diamino-pyrimidin-5- yloxy)-4-isopropyl-2-methoxy-phenyl]-3-ethyl-urea 21

5-(2,4-Diamino-pyrmidin-5-yloxy)- 4-isopropyl-2-methoxy-N-methyl-benzamide 22

1-[5-(2,4-Diamino-pyrimidin-5- yloxy)-4-isopropyl-2-methoxy-phenyl]-ethanol 23

5-(2,5-Diisopropyl-4-methoxy- phenoxy)-pyrimidine-2,4-diamine 24

5-[2-Isopropyl-4-methoxy-5-(1- methoxy-ethyl)-phenoxy]-pyrimidine-2,4-diamine 25

1-[5-(2,4-Diamino-pyrimidin-5- yloxy)-4-isopropyl-2-methoxy-phenyl]-3-phenyl-urea 26

5-(2,4-Diamino-pyrimidin-5-yloxy)- 4-isopropyl-2-methoxy-N-methyl-benzenesulfonamide 27

5-(2-Isopropyl-4-methoxy-5- trifluoromethoxy-phenoxy)-pyrimidine-2,4-diamine 28

5-(5-Iodo-2-isopropyl-4-prop-2- ynyloxy-phenoxy)-pyrimidine-2,4- diamine29

5-(2-Isopropyl-4-methoxy-5-nitro- phenoxy)-pyrimidine-2,4-diamine 30

5-(4-Ethoxy-5-iodo-2-isopropyl- phenoxy)-pyrimidine-2,4-diamine 31

5-[5-Iodo-5-isopropyl-4-(2,2,2- trifluoro-ethoxy)-phenoxy]-pyrimidine-2,4-diamine 32

5-(2-Isopropyl-4-methoxy-5-nitro- phenoxy)-pyrimidine-2,4-diamine 33

5-(5-Ethanesulfonyl-2-isopropyl-4- methoxy-phenoxy)-pyrimidine-2,4-diamine 34

5-(5-Fluoro-2-isopropyl-4-methoxy- phenoxy)-pyrimidine-2,4-diamine 35

2-[5-(2,4-Diamino-pyrimidin-5- yloxy)-4-isopropyl-2-methoxy-phenyl]-propan-2-ol 36

5-(2,4-Diamino-pyrimidin-5-yloxy)- N-ethyl-4-isopropyl-2-methoxy-benzenesulfonamide 37

5-(2,4-Diamino-pyrimidin-5-yloxy)- 4-isopropyl-2-methoxy-N,N-dimethyl-benzamide

As used in the specification and the appended claims, the singular forms“a”, “an,” and “the” include plural referents unless the context clearlydictates otherwise.

“Agonist” refers to a compound that enhances the activity of anothercompound or receptor site.

“Alkyl” means the monovalent linear or branched saturated hydrocarbonmoiety, consisting solely of carbon and hydrogen atoms, having from oneto twelve carbon atoms. “Lower alkyl” refers to an alkyl group of one tosix carbon atoms, e.g., C₁-C₆alkyl. Examples of alkyl groups include,but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl,sec-butyl, tert-butyl, pentyl, n-hexyl, octyl, dodecyl, and the like.

“Alkenyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbon atoms, containing at least one double bond, e.g., ethenyl,propenyl, and the like.

“Alkynyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbon atoms, containing at least one triple bond, e.g., ethynyl,propynyl, and the like.

“Alkylene” means a linear saturated divalent hydrocarbon radical of oneto six carbon atoms or a branched saturated divalent hydrocarbon radicalof three to six carbon atoms, e.g., methylene, ethylene,2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene,and the like.

“Alkoxy” means a moiety of the formula —OR, wherein R is an alkyl moietyas defined herein. Examples of alkoxy moieties include, but are notlimited to, methoxy, ethoxy, isopropoxy, and the like.

“Alkoxyalkyl” means a moiety of the formula R^(a)—O—R^(b)—, where R^(a)is alkyl and R^(b) is alkylene as defined herein. Exemplary alkoxyalkylgroups include, by way of example, 2-methoxyethyl, 3-methoxypropyl,1-methyl-2-methoxyethyl, 1-(2-methoxyethyl)-3-methoxypropyl, and1-(2-methoxyethyl)-3-methoxypropyl.

“Alkylcarbonyl” means a moiety of the formula —R′—R″, where R′ is (C═O)and R″ is alkyl as defined herein.

“Alkylsulfonyl” means a moiety of the formula —R′—R″, where R′ is —SO₂—and R″ is alkyl as defined herein.

“Alkylsulfonylalkyl” means a moiety of the formula —R′—R″—R′″ where R′is alkylene, R″ is —SO₂— and R′″ is alkyl as defined herein.

“Alkylamino” means a moiety of the formula —NR—R′ wherein R is hydrogenor alkyl and R′ is alkyl as defined herein.

“Alkylsulfanyl” means a moiety of the formula —SR wherein R is alkyl asdefined herein.

“Amino” means a moiety of the formula —NHR wherein R can be hydrogen oralkyl.

“Amido” means a moiety of the formula —NR(CO)R′— wherein R and R′ can beH or alkyl as defined herein.

“Hydroxy” means a moiety of the formula —OH.

“Haloalkoxy” means a group of the formula —OR, wherein R is a haloalkylgroup as defined herein.

“Nitro” means a group of the formula —NO₂. “Alkylcarbonyl” refers to agroup of the formula —(CO)R wherein R is an alkyl group as definedherein.

“Aminoalkyl” means a group —R—R′ wherein R′ is amino and R is alkyleneas defined herein. “Aminoalkyl” includes aminomethyl, aminoethyl,1-aminopropyl, 2-aminopropyl, and the like. The amino moiety of“aminoalkyl” may be substituted once or twice with alkyl to provide“alkylaminoalkyl” and “dialkylaminoalkyl” respectively.“Alkylaminoalkyl” includes methylaminomethyl, methylaminoethyl,methylaminopropyl, ethylaminoethyl and the like. “Dialkylaminoalkyl”includes dimethylaminomethyl, dimethylaminoethyl, dimethylaminopropyl,N-methyl-N-ethylaminoethyl, and the like.

“Aminosulfonyl” means a group —SO₂—NRR′ wherein R and R′ eachindependently is hydrogen or alkyl as defined herein.

“Alkylsulfonylamido” means a moiety of the formula —NR′SO₂—R wherein Ris alkyl and R′ is hydrogen or alkyl.

“Alkynylalkoxy” means a group of the formula —O—R—R′ wherein R isalkylene and R′ is alkynyl as defined herein.

“Aryl” means a monovalent cyclic aromatic hydrocarbon moiety consistingof a mono-, bi- or tricyclic aromatic ring. The aryl group can beoptionally substituted as defined herein. Examples of aryl moietiesinclude, but are not limited to, optionally substituted phenyl,naphthyl, phenanthryl, fluorenyl, indenyl, pentalenyl, azulenyl,oxydiphenyl, biphenyl, methylenediphenyl, aminodiphenyl,diphenylsulfidyl, diphenylsulfonyl, diphenylisopropylidenyl,benzodioxanyl, benzofuranyl, benzodioxylyl, benzopyranyl, benzoxazinyl,benzoxazinonyl, benzopiperadinyl, benzopiperazinyl, benzopyrrolidinyl,benzomorpholinyl, methylenedioxyphenyl, ethylenedioxyphenyl, and thelike, including partially hydrogenated derivatives thereof.

“Arylsulfonyl” means a group of the formula —SO₂—R wherein R is aryl asdefined herein.

“Antagonist” refers to a compound that diminishes or prevents the actionof another compound or receptor site.

“Cyanoalkyl” means a moiety of the formula —R′—R″, where R′ is alkyleneas defined herein and R″ is cyano or nitrile.

The terms “halo”, “halogen” and “halide”, which may be usedinterchangeably, refer to a substituent fluoro, chloro, bromo, or iodo.

“Haloalkyl” means alkyl as defined herein in which one or more hydrogenatoms have been replaced with the same or different halogen. Exemplaryhaloalkyls include —CH₂Cl, —CH₂CF₃, —CH₂CCl₃, perfluoroalkyl (e.g.,—CF₃), and the like.

“Hydroxyalkoxy” means a moiety of the formula —OR wherein R ishydroxyalkyl as defined herein.

“Hydroxyalkylamino” means a moiety of the formula —NR—R′ wherein R ishydrogen or alkyl and R′ is hydroxyalkyl as defined herein.

“Hydroxyalkylaminoalkyl” means a moiety of the formula —R—NR′—R″ whereinR is alkylene, R′ is hydrogen or alkyl, and R″ is hydroxyalkyl asdefined herein.

“Hydroxycarbonylalkyl” or “carboxyalkyl” means a group of the formula—R—(CO)—OH where R is alkylene as defined herein.

“Hydroxyalkyloxycarbonylalkyl” or “hydroxyalkoxycarbonylalkyl” means agroup of the formula —R—C(O)—O—R—OH wherein each R is alkylene and maybe the same or different.

“Hydroxyalkyl” means an alkyl moiety as defined herein, substituted withone or more, preferably one, two or three hydroxy groups, provided thatthe same carbon atom does not carry more than one hydroxy group.Representative examples include, but are not limited to, hydroxymethyl,2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl,1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl,4-hydroxybutyl, 2,3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl,2,3-dihydroxybutyl, 3,4-dihydroxybutyl and2-(hydroxymethyl)-3-hydroxypropyl

“Carboxy” means a group of the formula —O—C(O)—OH.

“Sulfonamido” means a group of the formula —SO₂—NR′R″ wherein R′, and R″each independently is hydrogen or alkyl.

“Optionally substituted”, for example when used with the term alkyl,means an alkyl group which is optionally substituted independently withone to three substituents, preferably one or two substituents selectedfrom any of the substituents defined herein, for instance: alkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, hydroxyalkyl, halo, nitro,cyano, hydroxy, alkoxy, amino, acylamino, mono-alkylamino,di-alkylamino, haloalkyl, haloalkoxy, heteroalkyl, —COR (where R ishydrogen, alkyl, phenyl or phenylalkyl), —(CR′R″)_(n)—COOR (where n isan integer from 0 to 5, R′ and R″ are independently hydrogen or alkyl,and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl orphenylalkyl), or —(CR′R″)_(n)—CONR^(a)R^(b) (where n is an integer from0 to 5, R′ and R″ are independently hydrogen or alkyl, and R^(a) andR^(b) are, independently of each other, hydrogen, alkyl, cycloalkyl,cycloalkylalkyl, phenyl or phenylalkyl).

“Leaving group” means the group with the meaning conventionallyassociated with it in synthetic organic chemistry, i.e., an atom orgroup displaceable under substitution reaction conditions. Examples ofleaving groups include, but are not limited to, halogen, alkane- orarylenesulfonyloxy, such as methanesulfonyloxy, ethanesulfonyloxy,thiomethyl, benzenesulfonyloxy, tosyloxy, and thienyloxy,dihalophosphinoyloxy, optionally substituted benzyloxy, isopropyloxy,acyloxy, and the like.

“Modulator” means a molecule that interacts with a target. Theinteractions include, but are not limited to, agonist, antagonist, andthe like, as defined herein.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not.

“Disease” and “Disease state” means any disease, condition, symptom,disorder or indication.

“Inert organic solvent” or “inert solvent” means the solvent is inertunder the conditions of the reaction being described in conjunctiontherewith, including for example, benzene, toluene, acetonitrile,tetrahydrofuran, N,N-dimethylformamide, chloroform, methylene chlorideor dichloromethane, dichloroethane, diethyl ether, ethyl acetate,acetone, methyl ethyl ketone, methanol, ethanol, propanol, isopropanol,tert-butanol, dioxane, pyridine, and the like. Unless specified to thecontrary, the solvents used in the reactions of the present inventionare inert solvents.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic, andneither biologically nor otherwise undesirable and includes that whichis acceptable for veterinary as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” of a compound means salts that arepharmaceutically acceptable, as defined herein, and that possess thedesired pharmacological activity of the parent compound. Such saltsinclude:

acid addition salts formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid,benzenesulfonic acid, benzoic, camphorsulfonic acid, citric acid,ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid,glutamic acid, glycolic acid, hydroxynaphtoic acid,2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid,malonic acid, mandelic acid, methanesulfonic acid, muconic acid,2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinicacid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, andthe like; or salts formed when an acidic proton present in the parentcompound either is replaced by a metal ion, e.g., an alkali metal ion,an alkaline earth ion, or an aluminum ion; or coordinates with anorganic or inorganic base. Acceptable organic bases includediethanolamine, ethanolamine, N-methylglucamine, triethanolamine,tromethamine, and the like. Acceptable inorganic bases include aluminumhydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate andsodium hydroxide.

The preferred pharmaceutically acceptable salts are the salts formedfrom acetic acid, hydrochloric acid, sulphuric acid, methanesulfonicacid, maleic acid, phosphoric acid, tartaric acid, citric acid, sodium,potassium, calcium, zinc, and magnesium.

It should be understood that any of the functional groups (e.g.,substituents) defined herein can be connected in any orientationrelative to the rest of the chemical structure. For instance, an amidogroup can be connected in either orientation: —R—(CO)NH—R′ or—R′(CO)NH—R.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same acid addition salt.

“Protective group” or “protecting group” means the group whichselectively blocks one reactive site in a multifunctional compound suchthat a chemical reaction can be carried out selectively at anotherunprotected reactive site in the meaning conventionally associated withit in synthetic chemistry. Certain processes of this invention rely uponthe protective groups to block reactive nitrogen and/or oxygen atomspresent in the reactants. For example, the terms “amino-protectinggroup” and “nitrogen protecting group” are used interchangeably hereinand refer to those organic groups intended to protect the nitrogen atomagainst undesirable reactions during synthetic procedures. Exemplarynitrogen protecting groups include, but are not limited to,trifluoroacetyl, acetamido, benzyl (Bn), benzyloxycarbonyl(carbobenzyloxy, CBZ), p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, tert-butoxycarbonyl (BOC), and the like. Theartisan in the art will know how to choose a group for the ease ofremoval and for the ability to withstand the following reactions.

“Solvates” means solvent additions forms that contain eitherstoichiometric or non-stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

“Sympathetic hyperactivity disorder” or “sympathetic disease” refers to,without limitation, hypertension, heart failure, stroke, and the like.“Carotid body hypertonicity disorders” include conditions where tonicdischarge of chemosensitive carotid sinus petrosal afferents hasdeveloped and drives an unfavorably imbalanced excessive excitatoryoutput of sympathetic tone to cardiovascular structures, for example, inhypertension, heart failure, chronic sleep apnea; and diseases in whichcommon symptoms are persistent and excessive breathlessness (dyspnea)and fatigue, which may include heart failure and chronic obstructivepulmonary disease (COPD).

The terms “those defined above” and “those defined herein” whenreferring to a variable incorporates by reference the broad definitionof the variable as well as preferred, more preferred and most preferreddefinitions, if any.

The terms “treating”, “contacting” and “reacting” when referring to achemical reaction means adding or mixing two or more reagents underappropriate conditions to produce the indicated and/or the desiredproduct. It should be appreciated that the reaction which produces theindicated and/or the desired product may not necessarily result directlyfrom the combination of two reagents which were initially added, i.e.,there may be one or more intermediates which are produced in the mixturewhich ultimately leads to the formation of the indicated and/or thedesired product.

“Primary hypertension,” also known as “essential hypertension” or“idiopathic hypertension” is the form of hypertension that bydefinition, has no identifiable cause.

“Secondary hypertension” or “inessential hypertension” is a type ofhypertension which by definition is caused by an identifiable underlyingsecondary cause. For instance, secondary hypertension can be caused byendocrine diseases, kidney diseases, and tumors. It also can be a sideeffect of many medications.

NOMENCLATURE AND STRUCTURES

In general, the nomenclature used in this Application is based onAUTONOM™ v.4.0; a Beilstein Institute computerized system for thegeneration of IUPAC systematic nomenclature. Chemical structures shownherein were prepared using ISIS® version 2.2. Any open valency appearingon a carbon, oxygen or nitrogen atom in the structures herein indicatesthe presence of a hydrogen atom.

All patents and publications identified herein are incorporated hereinby reference in their entirety.

In some embodiments, proof-of-concept experiments are carried out usinga test compound, e.g., Compound A:

Compound A is a benzyl-diaminopyrimidine P2X3 antagonist.

Methods

Certain aspects, embodiments and/or methods described herein are basedat least in part on the surprising finding that compounds of formula (I)have surprising affects on certain diseases and conditions such as, forexample, hypertension, heart failure, dyspnea, sleep apnea and the like.Accordingly provided herein include methods for treating such diseasesusing compounds of formula (1), including compounds of Table 1.

The methods provided herein include methods for treating a diseasemediated by a P2X3 or P2X2/3 receptor antagonist, said method comprisingadministering to a subject in need thereof an effective amount of acompound of formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomerthereof, wherein:

R¹ is hydrogen or optionally substituted C₁-C₆ alkyl;

R² is: alkyl; alkenyl; alkynyl; amino; aminosulfonyl; halo; amido;haloalkyl; alkoxy; hydroxy; haloalkoxy; nitro; hydroxyalkyl;alkoxyalkyl; hydroxyalkoxy; alkynylalkoxy; alkylsulfonyl; arylsulfonyl;carboxyalkyl; cyano or alkylcarbonyl.

Peripheral arterial chemosensation plays a critical role in thedetection of circulating levels of oxygen, carbon dioxide, pH and othermetabolites, allowing maintained homeostatic control of autonomicfunctions, ventilatory drive and cardiorespiratory control, andprotecting against acute deviations in these chemical stimuli. Theprimary chemosensory cells within the arterial system are glomus(type 1) cells of the carotid bodies, residing at the two carotidbifurcations. Glomus cell activation by reduced arterial O₂ or pH and/orelevated CO₂ increases communication to adjacent chemosensory afferentswithin the carotid sinus nerve via release of specific transmittersubstances (Fitzgerald et al., 2009, Adv. Exp. Med. Biol., 648: 19-28;Nurse, 2010, Exp. Physiol. 95(6):657-667).

Without wishing to be bound by theory, it is proposed that thesensitivity of this chemosensory mechanism becomes markedly heightenedin a variety of pathological conditions, leading to accentuated sensingof chemical changes as well as inappropriate spontaneous tonic discharge(hypertonicity); this sensitization leads to centrally mediated shiftsin the balance of autonomic outflow towards increased sympathetictransmission, and triggers common signs and symptoms of manycardiopulmonary and metabolic diseases including chronically increasedvascular resistance, cardiac output and blood pressure (Paton et al.,2013, Curr. Hypertens. Rep., 15:273-280), the perception of dyspnea andfatigue, and pro-inflammatory immune activation.

The identity of transmitter substances responsible for sensitizingcarotid sinus nerve excitability and contributing to tonic discharge hasbeen the subject of considerable work, with acetylcholine, dopamine,serotonin and ATP, among others, all suspected as participating (Nurse,2010, Exp. Physiol. 95(6):657-667: Kumar, 2007, Essays Biochem.43:43-60). In the case of ATP, it is released from chemosensitive glomuscells of the carotid body and is able to excite petrosal sensory nerveendings of the carotid sinus nerve (Zhang et al., 2000, J Physiol., 525:143-158; Prasad et al., 2001, J Physiol., 537: 667-677; Burnstock, 2009,Handb. Exp. Pharmacol. 194:333-92); recent evidence adds further supportby confirming the release of ATP in human carotid body tissues (Kåhlin Jet al., 2014 Exp Physiol (May 30) PMID: 24887113).

Without wishing to be bound by theory, it is proposed that theextracellular signaling functions of ATP result from activation of P2purinoceptors, which mediate many physiological and pathological roleson all types of cells (See, Burnstock (1993) Drug Dev. Res. 28:195-206).Among these signals, ATP is able to stimulate sensory nerve endings, inparticular on small and medium size sensory neurons, resulting in apronounced increase in sensory nerve discharge and the perception of avariety of unpleasant sensations such as pain, discomfort, fatigue,urinary urgency, itch, urge to cough and breathlessness, mediatedlargely via activation of receptors containing P2X3 subunits, onafferent nerve fibers innervating many rodent and human tissues(Burnstock, 2009, Handb. Exp. Pharmacol. 194:333-92; Ford and Undem,2013, Front Cell Neurosci. 7:267). These afferent nerve discharges alsovariously give rise to central modulation of efferent autonomic reflexessuch as for example, sympathetic activation of cardiovascular andimmunological structures or parasympathetic activation of urinary tracttissues.

Sensory innervation of the carotid body via the carotid sinus branch ofthe glossopharyngeal nerve arises from cells within the petrosal gangliathat express high concentrations of P2X3 and P2X2 receptor subunits,giving rise to excitatory ATP gated channels of the P2X3 homotrimericand P2X2/3 heterotrimeric forms (Prasad et al., 2001, J Physiol., 537:667-677). Accordingly, mice engineered as deletion mutants of eitherP2X3 or P2X2 subunits (P2X2KO and P2X3KO mice) or cross-bred doublegene-deletion mice (P2X2-P2X3dKO mice), show attenuated chemosensingresponses to ATP and hypoxia, as reflected by action potential firing ofthe carotid sinus nerve or plethysmographically-recorded ventilatoryresponses (Rong et al., 2003, J. Neurosci. 23(36):11315-11321).

Without wishing to be bound by theory, medicinal agents that selectivelymodulate P2X3 and P2X2/3 receptors by reducing their responsiveness tothe ATP released by carotid body glomus cells or other adjacent cells,or circulating in the blood are expected to reduce the level of sensorytransmission to carotid sinus afferents by attenuating the ATP-dependentcomponent, while leaving residual transmission to other non-purinergicsignals intact. Thereby the pattern of persistent tonic discharge andhypersensitivity seen in pathological conditions and other untowardsympathetically driven sequelae, can be broken, thus reducing theexcessive sympathetic outflow and reducing both excessive cardiac andvascular tone as well as perception of dyspnea and fatigue.Nevertheless, the presence of some signal redundancy (e.g., via ACh)will allow for the retention and preservation of important protectiveventilatory reflex responses, for example in the face of significanthypoxemia.

There is evidently a need for methods of treating diseases, conditionsand disorders mediated by P2X3 and/or P2X2/3 receptors, as well as aneed for compounds that act as modulators of P2X receptors, includingantagonists of P2X3 and P2X2/3 receptors. Such diseases and disordersare now, due to the work described herein, shown to include conditionsassociated with and largely resulting from enhanced sensitization of thecarotid body sensing mechanism, in particular where inappropriate tonicdischarge emanates within the carotid sinus nerve, as is reported tooccur in many clinical conditions and as described herein.

In various aspects and embodiments, provided herein include compoundsand methods for treatment of diseases driven by heightened sympatheticautonomic activity associated with P2X purinergic receptors, and moreparticularly to methods of using selective P2X3 and/or P2X2/3antagonists for treatment of common signs, symptoms and morbidity ofdiseases in which augmented carotid body chemosensory afferent dischargeand hypertonicity lead to the pathologically elevated sympathetic tonethat serves as a primary driver of such disorders.

The present disclosure further provides methods for treating diseases ofsympathetic nervous hyperactivity mediated by carotid body hypertonicityusing a P2X3 or P2X2/3 receptor antagonist. Increased nervous activityhas been shown to be directly correlated with hyperresponsiveness andtonic activity of the carotid sinus nerve in subjects, for instance inmammals such as humans or spontaneously hypertensive rats (SHRs). It canbe largely corrected by antagonism of P2X3-containing receptors (e.g.,P2X3 and P2X2/3). Moreover, the diaminopyrimidine antagonistsexemplified herein are highly effective at attenuating both the tonicdischarge of carotid sinus petrosal afferents, the sympathetic overactivity to cardiovascular structures, and the consequently elevatedblood pressure and ultimately associated end-organ damage. Theconditions, diseases, and disorders that are associated with carotidbody overactivity and tonic carotid sinus nerve discharge include, forexample, hypertension, chronic sleep apnea, insulin resistance and heartfailure.

Accordingly, in one aspect, the present technology may be directed tomethods for treating a subject having at least one symptom ofsympathetic hyperactivity and/or carotid body hypertonicity, comprisingadministering to a subject an effective amount of a P2X3 or P2X2/3receptor antagonist such as a compound of formula (I). In one or moreembodiments, the carotid body hypertonicity and/or sympathetichyperactivity is modulated by a P2X3 and/or a P2X2/3 receptorantagonist. Examples of diseases driven by heightened sympatheticautonomic activity associated with P2X purinergic receptors and/oraugmented carotid body chemosensory afferent discharge and hypertonicityinclude cardiovascular diseases and cardiopulmonary diseases asdescribed herein. The cardiovascular disease can be driven bysympathetic hyperactivity and can be selected from chronic hypertension,(e.g., treatment-resistant hypertension), chronic heart failure, acuteheart failure, arrhythmias, and stroke. The pulmonary condition can bedriven by sympathetic hyperactivity and can be selected from sleepdisordered breathing, and chronic obstructive sleep apnea. The pulmonarycondition can also be driven by carotid body hypertonicity and can beselected from dyspnea, breathlessness, or chest tightness. In someembodiments, the pulmonary condition (e.g., chest tightness) can beassociated with chronic obstructive pulmonary disease (COPD), heartfailure, peripheral artery disease, idiopathic pulmonary fibrosis, orother interstitial lung disease. The metabolic condition can be drivenby sympathetic hyperactivity and can be selected from diabetes mellitusand chronic kidney disease.

In another aspect, the present disclosure is directed to a method oftreating carotid body hypersensitivity, hypertonicity, or clinicalsequelae, in a subject by administering to such subject P2X3 or P2X2/3receptor antagonist such as a compound of formula (I). Exemplarydiseases of sympathetic nervous hyperactivity consequent to carotid bodyhypertonicity treatable with the invention include chronic hypertensionand heart failure, where effective, safe and well-tolerated medicalsuppression of sympathetic overdrive have been hitherto unachievable.For example, a disease treatable by the invention is chronic treatmentresistant hypertension, defined as persistent hypertension (restingoffice blood pressure>140/90 [SBP/DBP]) despite use of >3antihypertensive medications including a diuretic), as well ashypertension in patients unable to tolerate currently preferredantihypertensive medications, or in whom approved medications cannotachieve recommended levels of BP control.

A disease treatable in accordance with the compositions and methodsprovided herein can be chronic sleep apnea, where the effect ofrepetitive intermittent bouts of hypoxemia and hypercapnia directlycause increased tonic discharge of the carotid body and subsequentsympathetic hyperactivity, hypertension and cardiovascular pathology.

In one or more embodiments, the methods and compositions provided hereinare useful for a method for treating the signs and symptoms of carotidhypertonicity driving much of the cardiovascular morbidity associatedwith a cardiopulmonary disease, such as heart failure and chronicobstructive pulmonary disease, associated with pathological perceptionof fatigue, breathlessness, chest-tightness, dyspnea or the like.

In one or more embodiments, provided herein include a method oftreatment or prevention of stroke by reducing sympathetic overactivityby a P2X3 or P2X2/3 receptor antagonist, and lessening stress onhypocompliant, inflamed and friable vascular structures.

In one or more embodiments, provided herein include methods forimproving the signs and symptoms of insulin resistance, frequentlyassociated with diabetes mellitus; and slowing the progressivedeterioration in cardio-renal function in diabetic patients.

In another aspect, the administration of the compound of Formula I asdefined herein can be used for treating a subject having at least onesympathetically-mediated disease.

In another aspect, administration of the compound of Formula I asdefined herein can be used for suppressing the activity of the carotidbody in a subject. In one or more embodiments, the compounds of FormulaI can be used to treat (e.g., correct) aberrant discharge in the carotidbody.

In another aspect, administration of the compound of Formula I asdefined herein can be used for adjusting the autonomic balance in asubject having high sympathetic activity relative to parasympatheticactivity.

In another aspect, administration of the compound of Formula I asdefined herein can be used for inhibiting chemoreflex function generatedby a carotid body in a subject. In one or more embodiments, inhibitingchemoreflex function generated by a carotid body in a subject can beused to treat a disease, e.g., heart failure or hypertension.

The invention also provides pharmaceutical compositions and methods ofpreparing the same.

As put forth herein, the present technologies feature a class of P2X3and P2X2/3 antagonists for the treatment of diseases brought about byconditions including sympathetic nervous hyperactivity and carotid bodyhypertonicity. The present technology has the advantage of addressingthe root cause of these illnesses instead of merely treating symptomsassociated with the diseases. The technology also has the advantage ofleaving the carotid body intact and allowing its normal functioning totake place. Further features and advantages are set herein and will beapparent to one of skill in the art.

Diseases or disorders treatable by the present methods include those inwhich the carotid body (e.g., hypertonicity or hyperactivity of thecarotid body) plays a role. Exemplary diseases treatable with theinvention include chronic hypertension, treatment-resistanthypertension, heart-failure, chronic obstructive sleep apnea. Acondition treatable by the present methods includes chronically elevatedsympathetic activity, for example in hypertension and heart failure. Forexample, the invention relates to a method for treating the symptoms ofdyspnea, breathlessness and chest-tightness associated with acardiopulmonary or respiratory disease. For example, the presentdisclosure relates to a method of treatment of the stroke and diabetesmellitus associated with a sympathetic overactivity via treatment withby a P2X3 or P2X2/3 receptor antagonist.

In certain embodiments of the present disclosure the disease to betreated or prevented may be chronic carotid body hypertonicity. Forexample, the invention relates to methods for reducing blood pressure insubjects with inappropriately high sympathetic tone due to excessivesensitivity of arterial chemoreceptors.

In many embodiments of the present disclosure the disorder to be treatedor prevented is hypertension associated with a cardiopulmonary orcardiovascular disease.

Synthesis

Compounds of the present disclosure can be made by a variety of methods,and in some embodiments as depicted in the illustrative syntheticreaction schemes shown and described below. Syntheses of compounds foruse in the invention can also be performed according to teachingspresented in, for example, U.S. Pat. Nos. 8,524,725, 7,858,632,8,008,313; 8,003,788; 7,531,547; 7,741,484 and 7,799,796, each of whichis specifically incorporated herein in its entirety.

The starting materials and reagents used in preparing these compoundsgenerally are either available from commercial suppliers, such asAldrich Chemical Co., or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis; Wiley & Sons: New York,1991, Volumes 1-15; Rodd's Chemistry of Carbon Compounds, ElsevierScience Publishers, 1989, Volumes 1-5 and Supplementals; and OrganicReactions, Wiley & Sons: New York, 1991, Volumes 1-40. The followingsynthetic reaction schemes are merely illustrative of some methods bywhich the compounds of the present invention can be synthesized, andvarious modifications to these synthetic reaction schemes can be madeand will be suggested to one skilled in the art having referred to thedisclosure contained in this Application.

The starting materials and the intermediates of the synthetic reactionschemes can be isolated and purified if desired using conventionaltechniques, including but not limited to, filtration, distillation,crystallization, chromatography, and the like. Such materials can becharacterized using conventional means, including physical constants andspectral data.

Unless specified to the contrary, the reactions described hereinpreferably are conducted under an inert atmosphere at atmosphericpressure at a reaction temperature range of from about −78° C. to about150° C., more preferably from about 0° C. to about 125° C., and mostpreferably and conveniently at about room (or ambient) temperature,e.g., about 20° C.

Scheme A below illustrates a synthetic procedure usable to preparespecific compounds of formula (I) above, wherein R³, R⁴, R^(d), andR^(e) are as defined herein.

In step 1 of Scheme A, an O-alkylation is carried out by reaction ofphenol i with a haloacetonitrile such as iodoacetonitrile k, to affordcyano ether l. Numerous substituted phenols i are either commerciallyavailable or may be prepared by techniques well known in the art for usein step 1. For example, substituted aldehydes may be converted to thecorresponding phenols i via Baeyer-Villiger oxidation using peracid suchas mCPBA, as illustrated in the experimental examples below. Thealkylation of step 1 may be effected in the presence of mild base underpolar aprotic solvent conditions.

In step 2, a cyano enol ether compound n is formed by treatment of cyanoether l with a strong base such as sodium hydride, followed byintroduction of ester m to form an enolate (not shown), that in turn isO-alkylated by addition of iodomethane or other alkyl halide. This stepmay be carried out under polar aprotic solvent conditions.

In step 3 cyano enol ether n is reacted with guanidine compound o in thepresence of base, under polar aprotic conditions, to yielddiaminopyrimidine (p). The diaminopyrimidine (p) is a compound offormula (I) usable in the methods of the invention.

Numerous variations on the procedure of Scheme A are possible and willbe readily apparent to those skilled in the art.

Specific details for producing compounds of the invention are describedin the Examples below.

Use

The current technology provides compounds and methods that are useful intreating diseases in which modulation of the carotid body plays a role.For instance, the present technology provides compounds and methods toalleviate or reduce signs and/or symptoms of diseases that are causedand/or exacerbated by heightened sympathetic autonomic activity. In somepreferred embodiments, the heightened sympathetic autonomic activity isassociated with P2X purinergic receptors (e.g., P2X3 and/or P2X2/3receptors), and compounds of the current invention act as antagonists tothese receptors.

Exemplary diseases include cardiovascular (e.g., hypertension, heartfailure, stroke,) and metabolic diseases (e.g., diabetes mellitus).Other diseases include obstructive sleep apnea and arrhythmia.

Accordingly, in some embodiments, the invention provides methods fortreating a cardiovascular, cardiopulmonary or metabolic disease mediatedby excessive reactivity of the carotid body and carotid sinus nervesystem using an antagonist for ATP-gated receptors containing P2X3subunits (a P2X3 and/or P2X2/3 receptor antagonist), said methodcomprising administering to a subject in need thereof an effectiveamount of a compound of formula (I).

Exemplary cardiovascular or cardiopulmonary diseases where carotid bodyhypertonicity and associated sympathetic overactivity can be treatablewith the invention include hypertension, treatment resistanthypertension, heart failure, COPD, and chronic obstructive sleep apnea.A disease treatable by the invention includes treatment resistanthypertension, for example in subjects where the disease is eitheruncontrolled with existing antihypertensive options or intolerant tothem.

In certain embodiments of the invention the condition treatable by theinvention may be insulin resistance, such that progression of resistancetowards diabetes mellitus and related diseases could be delayed orforestalled.

Certain specific examples illustrated herein demonstrate the effect ofcompounds of certain embodiments of the present technology onsympathetic hyperactivity using a rat model. In some embodiments, theexamples set forth use Wistar rats, or spontaneously hypertensive rats(SHRs). The SHR is a well-validated model of human sympathetic overactivity and the pathogenesis of hypertension. As with humans, the SHRdevelops a hypersensitive and hypertrophic carotid body that precedesthe development of sympathoexcitation and hypertension. Accordingly, askilled artisan will understand that the Wistar and SHR model isappropriate for elucidating the effect of compounds of the presentinvention in a mammalian subject, such as a human patient. See, e.g.,Paton et al., Curr Hypertens Rep (2013) 15:273-280.

FIGS. 1A-1D show the effects of Compound A, a benzyl-diaminopyrimidineP2X3 antagonist which is seen to reduce carotid sinus nerve activity andexcitatory responses to hypoxia and ATP (or analogs), in an isolatedpreparation of carotid body/carotid sinus nerves. FIG. 1A shows thiseffect in a wild-type mouse P2X2-P2X2/3 carotid/sinus nerve preparation.As shown, the dual P2X3 and P2X2/3 antagonist Compound A attenuatesbasal tonic firing and hypoxia-induced excitation of carotid sinusafferents in an isolated carotid body preparation of mouse. In wild typepreparations, Compound A reversibly inhibits the response of sinus nervehypoxia. The IC₅₀ is in the range between 10-30 μM.

FIG. 1B shows the effects of Compound A on homotrimeric P2X3 mediatedresponses of the sinus nerve to α,β-MeATP and hypoxia in P2X2 KO mice.FIG. 1B demonstrates that the dual P2X3 and P2X2/3 antagonist Compound Aattenuates basal tonic firing and hypoxia, as well as α,β-MeATP inducedexcitation of carotid sinus afferents in an isolated carotid bodypreparation of P2X2 KO mouse. Greater potency is shown by effectivenessof lower concentrations of Compound A, consistent with the dominant roleof P2X3 homotrimers in the carotid sinus response in the absence of P2X2subunits.

FIG. 1C shows the effects of Compound A on homotrimeric P2X2 mediatedresponses of the sinus nerve to α,β-MeATP and hypoxia in P2X3 KO mice.FIG. 1C shows that the dual P2X2 and P2X2/3 antagonist Compound A failsto attenuate basal tonic firing and hypoxia or ATP-induced excitation ofcarotid sinus afferents in an isolated carotid body preparation of theP2X3 KO mouse. The non-selective antagonist PPADS did show inhibitoryactivity.

FIGS. 2A-D show the results of studies on the effects of P2X3 and P2X2/3antagonism on carotid body responses, carotid body hypertonicity,sympathetic discharge and blood pressure in spontaneously hypertensiverats.

FIG. 2A shows that in conscious, unrestrained spontaneously hypertensiverats, denervation of the carotid bodies bilaterally lowered botharterial pressure and renal sympathetic nerve activity (˜55%), an effectnot seen in normotensive Wistar rats (McBryde et al, 2013). These dataled to the hypothesis that there is aberrant carotid body activity inSHR that drives sympathetic activity. The term “renal symp” isunderstood to mean sympathetic nerve activity to the kidney.

FIG. 2B shows that spontaneously hypertensive rats show tonicity(aberrant discharge), depolarized membrane potential and exaggeratedfiring responses to chemoreceptor stimuli. There is an absence of tonicactivity in normotensive Wistar rats. Both the tonic activity in SHRsand the exaggerated chemical responses are markedly attenuated by acompound of formula (I), whereas Wistar rats are less affected. Blockadeof P2X3 receptors in the carotid body with a compound of formula (I)abolished tonic activity and suppressed peripheral chemoreceptor reflexevoke firing response in petrosal neurons in spontaneously hypertensiverats.

FIG. 2C shows the anti-hypertensive action of P2X3 receptor antagonism(bolus i.v.) in conscious, radio-telemetered spontaneously hypertensiverats is dependent on the integrity of carotid body chemoreceptors. Therewas no significant change in arterial pressure following P2X3 antagonismin normotensive Wistar rats (Pijacka, Ford & Paton, unpublished data).

FIG. 2D shows immunocytochemical staining for P2X3 receptors in thecarotid body of a hypertensive human subject. Glomus cells are labelledwith Nissl stain. The pattern of the P2X3 immunofluorescence resemblesthat of fiber bundles innervating clusters of glomus cells. Thisconfirms the expression of P2X3 and/or P2X2/3 receptors on targetsensory neurons.

In one or more embodiments, the present technology provides methods fortreating a respiratory disease mediated by a P2X3 or P2X2/3 receptorantagonist, the method comprising administering a compound of Formula I.Exemplary respiratory diseases include, but are not limited to chronicobstructive pulmonary disease (COPD), asthma, bronchospasm, and thelike.

In another embodiment, the present technology provides methods fortreating a subject suffering from a sympathetically-mediated diseasecomprising administering a compound of Formula I. Exemplarysympathetically-mediated diseases include, but are not limited toelevated sympathetic tone, an elevated sympathetic/parasympatheticactivity ratio, autonomic imbalance primarily attributable to centralsympathetic tone being abnormally high, or heightened sympathetic toneat least partially attributable to afferent excitation traceable tohypersensitivity or hyperactivity of a peripheral chemoreceptor (e.g.,carotid body). Other sympathetically mediated diseases include cardiac,metabolic, or pulmonary disease such as hypertension (e.g., refractoryhypertension), congestive heart failure (CHF), diabetes, periodbreathing, insulin resistance, heart failure, or dyspnea. In someembodiments, the hypertension is drug resistant hypertension, orhypertension resistant to renal denervation.

In one or more embodiments, the present technology provides methods forsuppressing the activity of the carotid body in a subject.

In one or more embodiments, the present technology provides methods foradjusting the autonomic balance in a subject having high sympatheticactivity relative to parasympathetic activity comprising administering acompound of Formula I. In some embodiments, this includes reducingsympathetic activity. In one or more embodiments, the present technologyprovides methods for inhibiting chemoreflex function generated by acarotid body comprising administering a compound of Formula I.

In one or more embodiments, compounds of the current disclosure can beused to inhibit the response of the sinus nerve in a subject to hypoxia.In some embodiments, the effect is dose-dependent. The response can alsobe reversible. In some embodiments, the IC₅₀ range is between about 1and 30 μm; about 10 and 30 μm; or between about 1 and 10 μm; or betweenabout 1 and 1000 nm, or between about 1 and 100 nm; or between about 100and 500 nm; or between about 500 and 1000 nm.

In some embodiments, compounds of the present disclosure can inhibit theresponse to α,β-meATP and hypoxia mediated by homomeric P2X3 receptors.

In some embodiments, compounds of the present disclosure can block ATPresponse in the sinus nerve of a subject (e.g., a human, or, forexample, a model of a human disease or disorder such as a P2X2 knockoutmouse). In some embodiments, the effect is reversible. In someembodiments, compounds of the present disclosure can reversibly blockhomomeric P2X3-mediated response of the sinus nerve to hypoxia.

In one or more embodiments, compounds of the present disclosure areselective antagonists of homomeric P2X3 and P2X2/3 receptors (e.g.,human or mouse homomeric P2X3 and P2X2/3 receptors). The IC₅₀ values forP2X3 inhibition can be about 3 μm (e.g., less than 3 μm). The IC₅₀values for P2X2/3 can be about 15 μm (e.g., less than 15 μm). In someembodiments, compounds of the present disclosure are more potent againstP2X3 than against P2X2/3 (e.g., 5× more potent, 10× more potent, 20×more potent, or 100× more potent). In some embodiments, the IC₅₀ rangeis between about 1 and 30 μm; about 10 and 30 μm; or between about 1 and10 μm; or between about 1 and 1000 nm, or between about 1 and 100 nm; orbetween about 100 and 500 nm; or between about 500 and 1000 nm.

In some embodiments, use of the compounds of the present disclosure toantagonize P2X3 can abolish carotid chemoreceptor tone but not reflexresponse. Compounds of the present disclosure in various aspects andembodiments disclosed herein can reduce the intrinsic excitability ofcarotid body petrosal cells in a subject (e.g., mammals includingrodents such as rats or mice, and primates such as apes or humans). Insome embodiments, the subjects (e.g., rats) are in a hyperoxiccondition.

Compounds of the present disclosure can decrease baseline andchemoreflex-induced sympathoexcitation in a subject (e.g., human or ahuman model such as SH rats). In some embodiments, the subjects (e.g.,humans or rats) are in a hyperoxemic or hypoxemic condition.

In some embodiments, the lowering of blood pressure mediated by P2X3receptor antagonism is dependent upon the carotid body. In someembodiments, the lowering of blood pressure takes place in a subject(e.g., a human or a rat).

Administration and Pharmaceutical Composition

The disclosure includes pharmaceutical compositions comprising at leastone compound of the present disclosure, or an individual isomer, racemicor non-racemic mixture of isomers or a pharmaceutically acceptable saltor solvate thereof, together with at least one pharmaceuticallyacceptable carrier, and optionally other therapeutic and/or prophylacticingredients.

In general, the compounds of the disclosure will be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. Suitable dosageranges are typically 1-500 mg daily, preferably 1-100 mg daily, and mostpreferably 1-30 mg daily, depending upon numerous factors such as theseverity of the disease to be treated, the age and relative health ofthe subject, the potency of the compound used, the route and form ofadministration, the indication towards which the administration isdirected, and the preferences and experience of the medical practitionerinvolved. One of ordinary skill in the art of treating such diseaseswill be able, without undue experimentation and in reliance uponpersonal knowledge and the disclosure of this Application, to ascertaina therapeutically effective amount of the compounds of the presentdisclosure for a given disease.

Compounds of the disclosure may be administered as pharmaceuticalformulations including those suitable for oral (including buccal andsub-lingual), rectal, nasal, topical, pulmonary, vaginal, or parenteral(including intramuscular, intraarterial, intrathecal, subcutaneous andintravenous) administration or in a form suitable for administration byinhalation or insufflation. The preferred manner of administration isgenerally oral using a convenient daily dosage regimen which can beadjusted according to the degree of affliction.

A compound or compounds of the disclosure, together with one or moreconventional adjuvants, carriers, or diluents, may be placed into theform of pharmaceutical compositions and unit dosages. The pharmaceuticalcompositions and unit dosage forms may be comprised of conventionalingredients in conventional proportions, with or without additionalactive compounds or principles, and the unit dosage forms may containany suitable effective amount of the active ingredient commensurate withthe intended daily dosage range to be employed. The pharmaceuticalcompositions may be employed as solids, such as tablets or filledcapsules, semisolids, powders, sustained release formulations, orliquids such as solutions, suspensions, emulsions, elixirs, or filledcapsules for oral use; or in the form of suppositories for rectal orvaginal administration; or in the form of sterile injectable solutionsfor parenteral use. Formulations containing about one (1) milligram ofactive ingredient or, more broadly, about 0.01 to about one hundred(100) milligrams, per tablet, are accordingly suitable representativeunit dosage forms.

The compounds of the disclosure may be formulated in a wide variety oforal administration dosage forms. The pharmaceutical compositions anddosage forms may comprise a compound or compounds of the presentdisclosure or pharmaceutically acceptable salts thereof as the activecomponent. The pharmaceutically acceptable carriers may be either solidor liquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. A solidcarrier may be one or more substances which may also act as diluents,flavouring agents, solubilizers, lubricants, suspending agents, binders,preservatives, tablet disintegrating agents, or an encapsulatingmaterial. In powders, the carrier generally is a finely divided solidwhich is a mixture with the finely divided active component. In tablets,the active component generally is mixed with the carrier having thenecessary binding capacity in suitable proportions and compacted in theshape and size desired. The powders and tablets preferably contain fromabout one (1) to about seventy (70) percent of the active compound.Suitable carriers include but are not limited to magnesium carbonate,magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,gelatine, tragacanth, methylcellulose, sodium carboxymethylcellulose, alow melting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as carrier, providing a capsule in which theactive component, with or without carriers, is surrounded by a carrier,which is in association with it. Similarly, cachets and lozenges areincluded. Tablets, powders, capsules, pills, cachets, and lozenges maybe as solid forms suitable for oral administration.

Other forms suitable for oral administration include liquid formpreparations including emulsions, syrups, elixirs, aqueous solutions,aqueous suspensions, or solid form preparations which are intended to beconverted shortly before use to liquid form preparations. Emulsions maybe prepared in solutions, for example, in aqueous propylene glycolsolutions or may contain emulsifying agents, for example, such aslecithin, sorbitan monooleate, or acacia. Aqueous solutions can beprepared by dissolving the active component in water and adding suitablecolorants, flavors, stabilizers, and thickening agents. Aqueoussuspensions can be prepared by dispersing the finely divided activecomponent in water with viscous material, such as natural or syntheticgums, resins, methylcellulose, sodium carboxymethylcellulose, and otherwell-known suspending agents. Solid form preparations include solutions,suspensions, and emulsions, and may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The compounds of the disclosure may be formulated for topicaladministration to the epidermis as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase and will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or coloring agents. Formulations suitable for topicaladministration in the mouth include lozenges comprising active agents ina flavored base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatine andglycerine or sucrose and acacia; and mouthwashes comprising the activeingredient in a suitable liquid carrier.

The subject compounds may be formulated for nasal administration. Thesolutions or suspensions are applied directly to the nasal cavity byconventional means, for example, with a dropper, pipette or spray. Theformulations may be provided in a single or multidose form. In thelatter case of a dropper or pipette, this may be achieved by the subjectadministering an appropriate, predetermined volume of the solution orsuspension. In the case of a spray, this may be achieved for example bymeans of a metering atomizing spray pump.

The compounds of the disclosure may be formulated for aerosoladministration, particularly to the respiratory tract and includingintranasal administration. The compound will generally have a smallparticle size for example of the order of five (5) microns or less. Sucha particle size may be obtained by means known in the art, for exampleby micronization. The active ingredient is provided in a pressurizedpack with a suitable propellant such as a chlorofluorocarbon (CFC), forexample, dichlorodifluoromethane, trichlorofluoromethane, ordichlorotetrafluoroethane, or carbon dioxide or other suitable gas. Theaerosol may conveniently also contain a surfactant such as lecithin. Thedose of drug may be controlled by a metered valve. Alternatively theactive ingredients may be provided in a form of a dry powder, forexample a powder mix of the compound in a suitable powder base such aslactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidine (PVP). The powder carrier will form agel in the nasal cavity. The powder composition may be presented in unitdose form for example in capsules or cartridges of e.g., gelatine orblister packs from which the powder may be administered by means of aninhaler.

When desired, formulations can be prepared with enteric coatings adaptedfor sustained or controlled release administration of the activeingredient. For example, the compounds of the present disclosure can beformulated in transdermal or subcutaneous drug delivery devices. Thesedelivery systems are advantageous when sustained release of the compoundis necessary and when subject compliance with a treatment regimen iscrucial. Compounds in transdermal delivery systems are frequentlyattached to a skin-adhesive solid support. The compound of interest canalso be combined with a penetration enhancer, e.g., Azone(1-dodecylazacycloheptan-2-one). Sustained release delivery systems areinserted subcutaneously into the subdermal layer by surgery orinjection. The subdermal implants encapsulate the compound in a lipidsoluble membrane, e.g., silicone rubber, or a biodegradable polymer,e.g., polylactic acid.

The pharmaceutical preparations are preferably in unit dosage forms. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

Exemplary pharmaceutical preparations for delivery by various routes areformulated as shown in the following Tables. “Active ingredient” or“Active compound” as used in the Tables means one or more of theCompounds of Formula I.

Composition for Oral Administration Ingredient % wt./wt. Activeingredient 20.0% Lactose 79.5% Magnesium stearate 0.5%

The ingredients are mixed and dispensed into capsules containing about100 mg each;

one capsule would approximate a total daily dosage.

Composition for Oral Administration Ingredient % wt./wt. Activeingredient 20.0% Magnesium stearate 0.5% Crosscarmellose sodium 2.0%Lactose 76.5% PVP (polyvinylpyrrolidine) 1.0%

The ingredients are combined and granulated using a solvent such asmethanol. The formulation is then dried and formed into tablets(containing about 20 mg of active compound) with an appropriate tabletmachine.

Composition for Oral Administration Ingredient Amount Active compound1.0 g Fumaric acid 0.5 g Sodium chloride 2.0 g Methyl paraben 0.15 gPropyl paraben 0.05 g Granulated sugar 25.5 g Sorbitol (70% solution)12.85 g Veegum K (Vanderbilt Co.) 1.0 g Flavoring 0.035 ml Colorings 0.5mg Distilled water q.s. to 100 ml

The ingredients are mixed to form a suspension for oral administration.

Parenteral Formulation Ingredient % wt./wt. Active ingredient 0.25 gSodium Chloride qs to make isotonic Water for injection 100

The active ingredient is dissolved in a portion of the water forinjection. A sufficient quantity of sodium chloride is then added withstirring to make the solution isotonic. The solution is made up toweight with the remainder of the water for injection, filtered through a0.2 micron membrane filter and packaged under sterile conditions.

Suppository Formulation Ingredient % wt./wt. Active ingredient 1.0%Polyethylene glycol 1000 74.5% Polyethylene glycol 4000 24.5%

The ingredients are melted together and mixed on a steam bath, andpoured into molds containing 2.5 g total weight.

Topical Formulation Ingredients Grams Active compound 0.2-2 Span 60 2Tween 60 2 Mineral oil 5 Petrolatum 10 Methyl paraben 0.15 Propylparaben 0.05 BHA (butylated hydroxy anisole) 0.01 Water q.s. 100

All of the ingredients, except water, are combined and heated to about60° C. with stirring. A sufficient quantity of water at about 60° C. isthen added with vigorous stirring to emulsify the ingredients, and waterthen added q.s. about 100 g.

Nasal Spray Formulations

Several aqueous suspensions containing from about 0.025-0.5 percentactive compound may in some embodiments be prepared as nasal sprayformulations. The formulations optionally contain inactive ingredientssuch as, for example, microcrystalline cellulose, sodiumcarboxymethylcellulose, dextrose, and the like. Hydrochloric acid may beadded to adjust pH. The nasal spray formulations may be delivered via anasal spray metered pump typically delivering about 50-100 microlitersof formulation per actuation. A typical dosing schedule is 2-4 spraysevery 4-12 hours.

Other suitable pharmaceutical carriers and their formulations aredescribed in Remington: The Science and Practice of Pharmacy 1995,edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton,Pa.

Non-Limiting List of Exemplary Embodiments

In addition to the aspects and embodiments described and providedelsewhere in this disclosure, the following non-limiting list ofparticular embodiments are specifically contemplated:

-   -   1. A method of treating hypertension in a subject, comprising        identifying a subject diagnosed with hypertension and        administering to the subject a compound of formula (I).    -   2. A method of treating heart failure in a subject, comprising        identifying a subject diagnosed with heart failure and        administering to the subject a compound of formula (I).    -   3. A method of treating dyspnea in a subject, comprising        identifying a subject diagnosed with dyspnea and administering        to the subject a compound of formula (I).    -   4. A method of treating sleep apnea in a subject, comprising        identifying a subject diagnosed with sleep apnea and        administering to the subject a compound of formula (I).    -   5. A method for altering carotid body tonicity or activity in a        subject, involving identifying a subject in need of altering        carotid body tonicity or activity and administering to the        subject a compound of formula (I).    -   6. A method for reducing carotid body tonicity in a subject,        comprising identifying a subject in need of reduction in carotid        body tonicity or activity and administering to the subject a        compound of formula (I).    -   7. A method of reducing carotid body chemosensory afferent        discharge in a subject, comprising identifying a subject in need        of reduction in carotid body chemosensory afferent discharge and        administering to the subject a compound of formula (I).    -   8. A method of treating a subject having at least one symptom of        sympathetic hyperactivity or carotid body hypertonicity, said        method comprising identifying a subject having at least one        symptom of sympathetic hyperactivity or carotid body        hypertonicity and administering to said subject an effective        amount of a compound of Formula (I); wherein the carotid body        hypertonicity or sympathetic hyperactivity is optionally        associated with cardiopulmonary, cardiovascular, or metabolic        disease.    -   9. A method of treating carotid body hypersensitivity,        hypertonicity, or clinical sequelae, in a subject, comprising        identifying a subject in need of carotid body hypersensitivity,        hypertonicity, or clinical sequelae treatment and administering        to said subject a compound of formula (1).    -   10. A method of suppressing the activity of the carotid body in        a subject, comprising identifying a subject in need of carotid        body activity suppression and administering to said subject a        compound of formula (1).    -   11. A method of adjusting the autonomic balance in a subject        having high sympathetic activity relating to parasympathetic        activity, comprising identifying an individual having high        sympathetic activity relating to parasympathetic activity, and        administering to said subject a compound of formula (I).    -   12. The method of any of the preceding embodiments wherein the        compound of formula (I) has below formula (I):

-   -    or a pharmaceutically acceptable salt thereof; wherein        -   R¹ is hydrogen or optionally substituted C₁-C₆ alkyl; and        -   R² is alkyl; alkenyl; alkynyl; amino; aminosulfonyl; halo;            amido; haloalkyl; alkoxy; hydroxy; haloalkoxy; nitro;            hydroxyalkyl; alkoxyalkyl; hydroxyalkoxy; alkynylalkoxy;            alkylsulfonyl; arylsulfonyl; carboxyalkyl; cyano or            alkylcarbonyl.    -   13. The method of any of the preceding embodiments, wherein R²        of the compound of formula (I) is haloalkyl, aminosulfonyl,        alkylsulfonyl alkylcarbonyl or carboxyalkyl.    -   14. The method of any of the preceding embodiments, wherein R²        of the compound of formula (I) is haloalkyl, and wherein the        alkyl is methyl.    -   15. The method of any of the preceding embodiments, wherein the        compound of formula (I) is selected from the group consisting of        compounds of Table 1.    -   16. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #1 on Table 1.    -   17. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #2 on Table 1.    -   18. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #3 on Table 1.    -   19. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #4 on Table 1.    -   20. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #5 on Table 1.    -   21. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #6 on Table 1.    -   22. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #7 on Table 1.    -   23. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #8 on Table 1.    -   24. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #9 on Table 1.    -   25. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #10 on Table 1.    -   26. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #11 on Table 1.    -   27. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #12 on Table 1.    -   28. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #13 on Table 1.    -   29. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #14 on Table 1.    -   30. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #15 on Table 1.    -   31. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #16 on Table 1.    -   32. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #17 on Table 1.    -   33. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #18 on Table 1.    -   34. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #19 on Table 1.    -   35. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #20 on Table 1.    -   36. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #21 on Table 1.    -   37. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #22 on Table 1.    -   38. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #23 on Table 1.    -   39. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #24 on Table 1.    -   40. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #25 on Table 1.    -   41. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #26 on Table 1.    -   42. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #27 on Table 1.    -   43. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #28 on Table 1.    -   44. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #29 on Table 1.    -   45. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #30 on Table 1.    -   46. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #31 on Table 1.    -   47. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #32 on Table 1.    -   48. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #33 on Table 1.    -   49. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #34 on Table 1.    -   50. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #35 on Table 1.    -   51. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #36 on Table 1.    -   52. The method of any of the preceding embodiments, wherein the        compound of formula (I) is Compound #37 on Table 1.    -   53. A method of treating hypertension in a subject, comprising        identifying a subject diagnosed with hypertension and        administering to the subject a compound of formula (I).    -   54. A method of treating heart failure in a subject, comprising        identifying a subject diagnosed with heart failure and        administering to the subject a P2X3 and/or a P2X2/3 receptor        antagonist.    -   55. A method of treating dyspnea in a subject, comprising        identifying a subject diagnosed with dyspnea and administering        to the subject a P2X3 and/or a P2X2/3 receptor antagonist.    -   56. A method of treating sleep apnea in a subject, comprising        identifying a subject diagnosed with sleep apnea and        administering to the subject a P2X3 and/or a P2X2/3 receptor        antagonist.    -   57. A method for altering carotid body tonicity or activity in a        subject, involving identifying a subject in need of altering        carotid body tonicity or activity and administering to the        subject a P2X3 and/or a P2X2/3 receptor antagonist.    -   58. A method for reducing carotid body tonicity in a subject,        comprising identifying a subject in need of reduction in carotid        body tonicity or activity and administering to the subject a        P2X3 and/or a P2X2/3 receptor antagonist.    -   59. A method of reducing carotid body chemosensory afferent        discharge in a subject, comprising identifying a subject in need        of reduction in carotid body chemosensory afferent discharge and        administering to the subject a P2X3 and/or a P2X2/3 receptor        antagonist.    -   60. A method of treating a subject having at least one symptom        of sympathetic hyperactivity or carotid body hypertonicity, said        method comprising identifying a subject having at least one        symptom of sympathetic hyperactivity or carotid body        hypertonicity and administering to said subject an effective        amount of a P2X3 and/or a P2X2/3 receptor antagonist; wherein        the carotid body hypertonicity or sympathetic hyperactivity is        optionally associated with cardiopulmonary, cardiovascular, or        metabolic disease.    -   61. A method of treating carotid body hypersensitivity,        hypertonicity, or clinical sequelae, in a subject, comprising        identifying a subject in need of carotid body hypersensitivity,        hypertonicity, or clinical sequelae treatment and administering        to said subject a P2X3 and/or a P2X2/3 receptor antagonist.    -   62. A method of suppressing the activity of the carotid body in        a subject, comprising identifying a subject in need of carotid        body activity suppression and administering to said subject a        P2X3 and/or a P2X2/3 receptor antagonist.    -   63. A method of adjusting the autonomic balance in a subject        having high sympathetic activity relating to parasympathetic        activity, comprising identifying an individual having high        sympathetic activity relating to parasympathetic activity, and        administering to said subject a P2X3 and/or a P2X2/3 receptor        antagonist.    -   64. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a systolic        blood pressure above about 120 mmHg and/or a diastolic pressure        above about 80 mmHg.    -   65. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a systolic        blood pressure above about 125 mmHg.    -   66. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a systolic        blood pressure above about 130 mmHg.    -   67. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a systolic        blood pressure above about 140 mmHg.    -   68. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a systolic        blood pressure above about 150 mmHg.    -   69. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a systolic        blood pressure above about 160 mmHg.    -   70. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a diastolic        pressure above about 85 mmHg.    -   71. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a diastolic        pressure above about 90 mmHg.    -   72. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a diastolic        pressure above about 95 mmHg.    -   73. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, has a diastolic        pressure above about 100 mmHg.    -   74. The method of any of the preceding embodiments, wherein a        subject diagnosed with hypertension, if present, is diagnosed        with chronic treatment resistant hypertension.    -   75. The method of any of the preceding embodiments, wherein said        subject diagnosed with heart failure, if present, is diagnosed        with systolic heart failure.    -   76. The method of any of the preceding embodiments, wherein said        subject diagnosed with heart failure, if present, is diagnosed        with diastolic heart failure.    -   77. The method of any of the preceding embodiments, wherein said        subject diagnosed with heart failure, if present, is diagnosed        with chronic heart failure.    -   78. The method of any of the preceding embodiments, wherein said        subject diagnosed with heart failure, if present, is diagnosed        with acute heart failure.    -   79. The method of any of the preceding embodiments, wherein said        subject diagnosed with sleep apnea, if present, is diagnosed        with central sleep apnea.    -   80. The method of any of the preceding embodiments, wherein said        subject diagnosed with sleep apnea, if present, is diagnosed        with obstructive sleep apnea.    -   81. The method of any of the preceding embodiments, wherein said        subject diagnosed with sleep apnea, if present, is diagnosed        with mixed sleep apnea.    -   82. The method of any of the preceding embodiments, wherein said        subject diagnosed with sleep apnea, if present, is diagnosed as        having more than about 5 apneic events per hour of sleep.    -   83. The method of any of the preceding embodiments, wherein said        subject diagnosed with sleep apnea, if present, is diagnosed as        having more than about 15 apneic events per hour of sleep.    -   84. The method of any of the preceding embodiments, wherein said        subject diagnosed with sleep apnea, if present, is diagnosed as        having more than about 25 apneic events per hour of sleep.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice certaintechnologies, methods, inventions, compositions, concepts disclosedherein. They should not be considered as limiting the scope of anyinventions, but merely as being illustrative and representative thereof.

Example 15-(5-Bromo-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine

The synthetic procedure used in this Example is outlined in Scheme B.

Step 1. 2-Bromo-5-isopropyl-phenol

A solution of 3-isopropyl phenol (4.975 g, 36.5 mmol) in 37 mL of CCl₄was cooled to −20° C. Bromine (1.9 mL, 38.4 mmol) was dissolved in 5.0mL CCl₄ and added drop-wise at such a rate that the internal temperaturewas maintained below −10° C. The mixture was allowed to warm to roomtemperature. After 12 hours the mixture was taken up in 100 mL CH₂Cl₂,washed with H₂O and then with brine. The combined organics were driedover Na₂SO₄, filtered and concentrated in vacuo to give 8.663 g of a 1:1mixture of 2-bromo-5-isopropyl-phenol and 4-bromo-5-isopropyl phenol asa dark oil). These two isomers were inseparable and were used togetherin step 2 below.

Step 2. 1-Bromo-4-isopropyl-2-methoxy-benzene

To a mixture of 2-bromo-5-isopropyl-phenol and 4-bromo-5-isopropylphenol from step 1 (8.663 g, 40.3 mmol), K₂CO₃ (16.710 g, 120.9 mmol) in50 mL DMF, was added iodomethane (3.0 mL, 48.3 mmol) with mechanicalstirring. The mixture was warmed to 50° C. for 4 hours. After cooling toroom temperature 300 mL H₂O was added and the solution was extractedwith diethyl ether (Et₂O), washed with H₂O and washed with brine. Thecombined organics were dried over MgSO₄, filtered and concentrated invacuo to give 1-bromo-4-isopropyl-2-methoxy-benzene and1-bromo-2-isopropyl-4-methoxy-benzene (6.621 g, 72%) as a 1:1inseparable mixture in the form of a pale yellow oil. This mixture ofregioisomers was used directly in step 3 below.

Step 3. 5-Bromo-2-isopropyl-4-methoxy-benzaldehyde

To a solution of 1-bromo-4-isopropyl-2-methoxy-benzene and1-bromo-2-isopropyl-4-methoxy-benzene from step 2 (6.621 g, 28.9 mmol)in 100 mL 1,2 dichloroethane was added TiCl₄ (6.3 mL, 57.8 mmol) at 0°C. After 10 minutes, dichloromethoxymethane (Cl₂CHOMe) (2.6 mL, 28.9mmol) was added and the mixture was warmed to reflux. After 3 hours themixture was cooled poured over ice and acidified with 50 mL 2 M HCl. Theresulting slurry was extracted with CH₂Cl₂, and washed with brine. Thecombined organics were dried over MgSO₄, filtered and concentrated invacuo to give a dark-green oil. Purification via flash chromatography(96:4 hexane/ethyl acetate) afforded5-bromo-2-isopropyl-4-methoxy-benzaldehyde and5-bromo-4-isopropyl-2-methoxy-benzaldehyde (2.876 g, 39%, 6.621 g, 72%)as a 1:1 mixture of inseparable isomers in the form of an orange oil,which was used directly in step 4.

Step 4. 5-Bromo-2-isopropyl-4-methoxy-phenol

To a solution of 5-bromo-2-isopropyl-4-methoxy-benzaldehyde and5-bromo-4-isopropyl-2-methoxy-benzaldehyde from step 3 (2.87 g, 11.2mmol) in 25 mL CH₂Cl₂ was added mCPBA (2.31 g, 13.4 mmol). After 16hours the mixture was taken up in 150 ml CH₂Cl₂ and washed with satNaHCO₃, and then with brine. The combined organic layers were dried overNa₂SO₄, filtered and concentrated in vacuo to give an oil that was takenup in 50 mL MeOH and 30 mL 4M NaOH. After 2 hours the mixture wasevaporated, diluted with water and acidified to pH=1 with concentratedHCl. The mixture was extracted with ethyl acetate (3×100 mL) and washedwith 100 mL brine. The combined organics were dried over Na₂SO₄,filtered and evaporated to give a mixture of5-bromo-2-isopropyl-4-methoxy-phenol and2-bromo-5-isopropyl-4-methoxy-phenol as an orange residue. Theseregioisomers were separable by flash chromatography (gradient: hexane,7:3, 1:1 hexane/CH₂Cl₂) to afford 5-bromo-2-isopropyl-4-methoxy-phenol(0.929, 34%) as a yellow oil which was used in the following step, and2-bromo-5-isopropyl-4-methoxy-phenol (0.404 g, 15%) as a yellow solid.

Step 5. (5-Bromo-2-isopropyl-4-methoxy-phenoxy)-acetonitrile

To a mixture of 5-bromo-2-isopropyl-4-methoxy-phenol from step 4 (0.831g, 3.4 mmol) and K₂CO₃ (0.562 g, 4.1 mmol) in 17 mL dimethyl formamide(DMF) was added iodoacetonitrile (0.594 g, 3.6 mmol). The mixture waswarmed to 60° C. for 30 minutes and then allowed to cool to roomtemperature. After cooling to room temperature the mixture was taken upin 50 mL of H₂O and extracted with 1:1 toluene/ethyl acetate, washedwith H₂O and then with brine. The combined organic layers were driedover Na₂SO₄, filtered and concentrated in vacuo to give a crude solid.Purification via flash chromatography (1:1 hexane/CH₂Cl₂) afforded(5-bromo-2-isopropyl-4-methoxy-phenoxy)-acetonitrile (0.611 g, 63%) as awhile solid.

Step 6.2-(5-Bromo-2-isopropyl-4-methoxy-phenoxy)-3-methoxy-acrylonitrile

Sodium hydride (0.122 g, 5.0 mmol, 60% w/w) was washed with dry hexanesand evaporated under a stream of nitrogen. 10 mL THF was added and themixture was cooled to 0° C.(5-Bromo-2-isopropyl-4-methoxy-phenoxy)-acetonitrile (0.577 g, 2.03mmol) was added in portions. After 30 min ethyl formate (4.9 mL, 60.9mmol) was added and the solution was warmed to 80° C. After 4.5 hoursthe mixture was cooled and 5.0 mL iodomethane was added in one portion.After 16 hours the solution was quenched with H₂O, concentrated invacuo, extracted with ethyl acetate, washed with H₂O and then washedwith brine. The combined organic layers were dried over Na₂SO₄, filteredand concentrated in vacuo. Purification via flash chromatography (9:1hexane/ethyl acetate) afforded2-(5-bromo-2-isopropyl-4-methoxy-phenoxy)-3-methoxy-acrylonitrile (0.319g, 48%) as a white solid.

Step 7. 5-(5-Bromo-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine

To a solution of2-(5-bromo-2-isopropyl-4-methoxy-phenoxy)-3-methoxy-acrylonitrile (0.282g, 0.9 mmol) and guanidine carbonate (0.078 g, 0.4 mmol) in 10.0 mLdimethyl sulfoxide (DMSO) was added sodium methoxide (1.0 mL, 1.0M inMeOH). The mixture was warmed to 120° C. The methanol was collected viaa short-path condenser. After 3 h the mixture was cooled andconcentrated in vacuo to give a crude oil. Purification via flashchromatography (95:5 CH₂Cl₂/MeOH) afforded 17 (0.246 g, 77%) as a pinksolid; Mass Spec M+H=352. The above procedure may be used with variousdifferent phenols in step 1 and/or substituted guanidines in step 7under essentially the same reaction conditions to produce additionalcompounds. Additional compounds made according to the procedure ofExample 1 are shown in Table 1.

Example 25-(2-Isopropyl-5-methanesulfonyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine

To a mixture of 5-(2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine(0.32 g, 1.17 mmol), prepared according to Example 1, andmethanesulfonic anhydride (0.81 g, 4.67 mmol) was addedtrifluoromethanesulfonic acid (0.45 g, 3.00 mmol), and the mixture washeated at 80° C. for 16 hrs. The reaction mixture was poured into icewater, basified with saturated NaHCO₃ solution and extracted intodichloromethane, which was dried over Na₂SO₄, filtered and concentratedin vacuo. The residue was purified via flash chromatography on silicagel (3% CH₃OH in CH₂Cl₂ with 0.1% NH₄OH) gave5-(2-isopropyl-5-methanesulfonyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamineas a white solid (0.248 g, 90%; 0.107 g), MS (M+H): 353.

Example 35-(5-Iodo-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine

To a solution of5-(2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (0.40 g, 1.44mmol) in glacial acetic acid (4 ml) at room temperature was added asolution of iodine monochloride (0.28 g, 1.76 mmol) in glacial aceticacid (4 ml). Water (6 ml) was also added, and the reaction was stirredfor 16 hours, after which another portion of iodine monochloride (0.4 g,2.47 mmol) in glacial acetic acid (4 ml) was added. The reaction mixturewas stirred for an additional hour at room temperature. The acidicmixture was basified with saturated NaHCO₃ solution and extracted intodichloromethane. The organic layer was dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was purified via flash chromatography(5% CH₃OH in CH₂CL₂ with 0.1% NH₄OH) to give5-(5-iodo-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine as beigecolored solid (0.536 g, 92%). M+H 400.

Example 45-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzonitrile

A mixture of5-(5-iodo-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (0.37 g,0.925 mmol) and CuCN (0.12 g, 1.39 mmol) in DMF (5 ml) was heated at120° C. for 3 hours. Water (100 ml) was added, and the precipitate wascollected. The residue was triturated with methanolic dichloromethane(10% CH₃OH in CH₂Cl₂ with 0.1% NH₄OH) to release the product from itscopper complex and filtered. The filtrate was concentrated and purifiedvia flash chromatography (3% CH₃OH in CH₂Cl₂ with 0.1% NH₄OH) to give5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzonitrile aswhite solid (0.12 g, 44%): M+H 300.

Example 51-[5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-ethanoneand1-[5-(2,4-Diamino-pyrimidin-5-yloxy)-2-hydroxy-4-isopropyl-phenyl]-ethanone

5-(2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine in anhydrousdichloroethane (20 mL) was added to trifluoroacetic acid (0.06 mL, 0.77mmol), acetyl chloride (0.31 mL, 4.37 mmol), and aluminum trichloride(583 mg, 4.37 mmol). After stirring for 22 hours at room temperature,water (1.2 mL) was added to the reaction at 0° C. The mixture was driedusing anhydrous sodium sulfate and concentrated in vacuo. Aqueous sodiumhydroxide (0.2M, 10 mL) was added to the residue and the mixture washeated at 100° C. for 1 hour. After cooling, the reaction was extractedwith dichloromethane. The dichloromethane layer was dried usinganhydrous magnesium sulfate, concentrated, and purified with silica gelcolumn chromatography eluting with 96/4/0.1dichloromethane/methanol/ammonium hydroxide to yield1-[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-ethanone(72 mg, 31%) as off-white solid, MS (M+H)=317. Also recovered was1-[5-(2,4-diamino-pyrimidin-5-yloxy)-2-hydroxy-4-isopropyl-phenyl]-ethanone(43 mg, 20%) as pale yellow solid, MS (M+H)=303.

Example 65-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzoic acid

To a suspension of5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzonitrile (50mg, 0.17 mmol, from Example 14) in ethanol (1 mL) was added sodiumhydroxide (174 mg, 4.34 mmol, dissolved in 1 mL water). After refluxingovernight, the reaction was cooled in an ice bath. Aqueous hydrochloricacid (3M) was added until the pH of the reaction was 7. The white solidprecipitate was collected, washed with small amounts of water anddichloromethane, and dried to yield5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzoic acid:(51 mg, 96%, MS (M+H)=319), which was converted to the hydrochloridesalt.

Example 75-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzamide

To 5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzonitrile(49 mg, 0.16 mmol, from Example 14) suspended in ethanol (1 mL) wasadded sodium hydroxide (64 mg, 1.60 mmol, dissolved in 1 mL water). Thereaction was heated at 110° C. for 5 hours, cooled, and washed withdichloromethane (25 mL). The dichloromethane layer was concentrated andpurified by preparatory TLC plates (92/8/0.5dichloromethane/methanol/ammonium hydroxide) to yield5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzamide aswhite solid (9 mg, 17%, MS (M+H)=318), which was converted to thehydrochloride salt.

Example 85-(2-Isopropyl-4-methoxy-5-nitro-phenoxy)-pyrimidine-2,4-diamine

The synthetic procedure used in this Example is outlined in Scheme C

Step 1. 2-(1-Hydroxy-1-methyl-ethyl)-4-methoxy-phenol

To a solution of methylmagnesium bromide (221 ml, 665 mmol) in 800 mlTHF at 0° C. was added 1-(2-hydroxy-5-methoxy-phenyl)-ethanone (20.21 g,302 mmol) in portions over 30 min. The mixture was allowed to warm toroom temperature. After 16 h the mixture was quenched by the slowaddition of 10% NH₄Cl, carefully acidified to pH=1 (slow addition) withconcentrated HCl and extracted with Et₂O. The combined organics werewashed with H₂O, washed with brine, died over MgSO₄, filtered andconcentrated in vacuo to give2-(1-hydroxy-1-methyl-ethyl)-4-methoxy-phenol (50.57 g, 100%) as a tansolid.

Step 2. 2-Isopropyl-4-methoxy-phenol

To a solution of 2-(1-hydroxy-1-methyl-ethyl)-4-methoxy-phenol (50.57 g,278 mmol) in 550 ml AcOH was added 10% Pd/C (as a slurry in 20 ml H₂O).Ammonium formate (87.52 g, 1388 mmol) was added in portions. The mixturewas warmed to 100° C. for 1 hour, cooled and filtered through a pad ofcelite. The celite pad was washed with ethyl acetate. The mother liquorwas mixed with H₂O and extracted with ethyl acetate. The combinedorganics were washed with H₂O, washed with brine, dried over Na₂SO₄,filtered and concentrated in vacuo to give 2-isopropyl-4-methoxy-phenol(44.74 g, 97%) as a pale yellow oil.

Step 3. Toluene-4-sulfonic acid 2-isopropyl-4-methoxy-phenyl ester

A solution of 2-isopropyl-4-methoxy-phenol (56.91 g, 342 mmol)triethylamine (57.3.0 ml, 411 mmol) in 750 ml CH₂Cl₂ was cooled to 0° C.p-Toluenesulfonyl chloride (68.54 g, 360 mmol) in 250 ml CH₂Cl₂ wasadded drop-wise at a rate that maintained the internal temperature<10°C. The mixture was allowed to warm to rt. After 16 h, H₂O was added andthe mixture was extracted with CH₂Cl₂. The combined organics were washedwith brine, dried with Na₂SO₄, filtered and concentrated in vacuo toafford a crude solid. Recrystallization from hexanes affordedtoluene-4-sulfonic acid 2-isopropyl-4-methoxy-phenyl ester (81.67 g,74%) as white needles.

Step 4. Toluene-4-sulfonic acid 2-isopropyl-4-methoxy-5-nitro-phenylester

To a solution of toluene-4-sulfonic acid 2-isopropyl-4-methoxy-phenylester (19.00 g, 59 mmol) in 118 mL AcOH was added 236 ml fuming HNO₃over 20 min. After 16 h the solution was pouring into a rapidly stirringslurry of 21 of ice/H₂O. After 15 min the precipitate was filtered,washed with H₂O and dried under vacuum (50° C.) to givetoluene-4-sulfonic acid 2-isopropyl-4-methoxy-5-nitro-phenyl ester(21.27 g, 98%) and toluene-4-sulfonic acid2-isopropyl-4-methoxy-3-nitro-phenyl ester and as a pale yellow solid(7:1 inseparable mixture).

Step 5. 2-Isopropyl-4-methoxy-5-nitro-phenol

A solution of toluene-4-sulfonic acid2-isopropyl-4-methoxy-5-nitro-phenyl ester and2-isopropyl-4-methoxy-3-nitro-phenyl ester (21.20 g, 58 mmol) and 175 mL2M KOH in 350 mL EtOH was warmed to 100° C. After 45 minutes the mixturewas cooled, evaporated and taken up in 1 l of water. The solution wasacidified to pH=1 with 12 M HCl and extracted with ethyl acetate. Thecombined organics were washed with H₂O, brine, dried over Na₂SO₄,filtered and concentrated in vacuo. The crude oil was purified via flashchromatography (gradient: 95:5 to 4:1 hexane/ethyl acetate) to afford3-amino-2-isopropyl-5-nitro-phenol (10.03 g, 81%) as a yellow solid and3-amino-2-isopropyl-3-nitro-phenol (1.32 g, 11%) as a yellow oil.

Step 6. (2-Isopropyl-4-methoxy-5-nitro-phenoxy)-acetonitrile

A mixture of 3-amino-2-isopropyl-5-nitrophenol (9.94 g, 47 mmol), K₂CO₃(13.00 g, 94 mmol) and toluenesulfonic acid cyanomethyl ester (10.93 g,52 mmol) in 500 mL DMF was warmed to 50° C. After 16 h the mixture wascooled, poured into 500 mL H₂O and extracted with toluene/ethyl acetate(1:1). The combined organics were washed with H₂O, washed with brine,filtered and concentrated in vacuo. The crude solid was recrystallizedfrom EtOH to afford (2-isopropyl-4-methoxy-5-nitro-phenoxy)-acetonitrile(8.95 g, 76%) as a yellow crystalline solid.

Step 7. 5-(2-Isopropyl-4-methoxy-5-nitro-phenoxy)-pyrimidine-2,4-diamine

A mixture of (2-isopropyl-4-methoxy-5-nitro-phenoxy)-acetonitrile (8.785g, 35.5 mmol) and Bredereck's reagent (14.6 mL, 70.9 mmol) was warmed to100° C. After 45 min the mixture was evaporated under reduced pressure(50° C., 50 mtorr) to give an orange solid. The solid was added to asolution of aniline hydrochloride (9.19 g, 70.9 mmol) in 150 mL of EtOH.The mixture was warmed to reflux. After 16 hr additional anilinehydrochloride (4.596 g, 35.5 mmol) was added mixture was continued atreflux for 4 h. The solution was concentrated in vacuo and poured intoH₂O. The mixture was extracted with ethyl acetate, washed with H₂O,washed with brine, dried over Na₂SO₄, and concentrated in vacuo toafford a yellow-green solid. This crude product was added to a mixtureof 200 mL NMP and guanidine carbonate (17.70 g, 98 mmol) and warmed to130° C. After 5 hours the mixture was cooled then poured onto 21 of anice/H₂O mixture. The resulting precipitate was filtered, washed with H₂Oand dried under vacuum (50° C.). The crude solid was recrystallized fromEtOH to afford5-(2-isopropyl-4-methoxy-5-nitro-phenoxy)-pyrimidine-2,4-diamine (8.14g, 63%, 3 steps) as a yellow crystalline solid (solvated 1:1 with EtOH).(M+H)⁺=320.

Example 9[5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-ureaStep 1. 5-(5-Amino-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine

To 5-(2-isopropyl-4-methoxy-5-nitro-phenoxy)-pyrimidine-2,4-diamine (2.1g, 6.58 mmol) suspended in ethanol (150 mL) in a Parr bomb, was added10% palladium on charcoal (210 mg). After hydrogenation in the Parrhydrogenator overnight at 35 psi, the reaction was filtered throughcelite. The celite pad was washed with ethanol and ethyl acetate and thefiltrate was concentrated. Purification with silica gel columnchromatography (92/8/0.1 dichloromethane/methanol/ammonium hydroxide)gave 5-(5-amino-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine asa pale orange solid (468 mg, 25%, (M+H)⁺=290), which was converted tothe hydrochloride salt.

Step 2.[5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-urea

To 5-(5-amino-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (314mg, 1.09 mmol) suspended in water (3 mL) was added acetic acid (0.25 mL,4.34 mmol). Once all solids had dissolved, sodium cyanate (71 mg, 1.09mmol, dissolved in 1.5 mL water) was added dropwise. After 30 minutes,the reaction was concentrated and purified with silica gel columnchromatography eluting with 92/8/0.1 dichloromethane/methanol/ammoniumhydroxide to yield[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-urea asan off-white solid (244 mg, 68%, M+H)⁺=333), which was converted to ahydrochloride salt:

Example 10N-[5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-acetamide

To 5-(5-amino-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (100mg, 0.35 mmol, from Example 17) dissolved in anhydrous dichloromethane(10 mL) was added anhydrous pyridine (0.03 mL, 0.38 mmol). To thisreaction mixture at 0° C. was added acetyl chloride (0.03 mL, 0.38mmol). After stirring at room temperature for 1 hour, the reaction wasconcentrated and purified with preparatory TLC (93/7/0.5dichloromethane/methanol/ammonium hydroxide) to yield an off-white solid(74 mg mixture of bis- and tris-acetylated products). To this solid wasadded aqueous sodium hydroxide (0.2 M, 2 mL), and the mixture wasrefluxed for 1 hour, cooled, and washed with dichloromethane (10 mL).The dichloromethane layer was dried using anhydrous magnesium sulfateand concentrated in vacuo to yieldN-[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-acetamideas a white solid (53 mg, 46%, M+H)⁺=332) which was converted to ahydrochloride salt:

Example 111-[5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-3-ethyl-ureaStep 1. 5-(5-Amino-2-Isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine

To a solution of5-(2-isopropyl-4-methoxy-5-nitro-phenoxy)-pyrimidine-2,4-diamine (2.953g, 9.2 mmol) in 250 mL EtOH and 25 AcOH was added 10% Pd/C. The mixturewas placed under 50 psi of H₂ via a Parr hydrogenator. After 2.5 h themixture was filtered through a pad of celite. The pad was washed withethyl acetate and the solution was partially concentrated in vacuo. Theresidue was taken up in 500 mL H₂O and cooled to 0° C. The solution wasadjusted to pH=12 with 50% NaOH extracted with ethyl acetate. Thecombined organics were washed with H₂O, washed with brine, dried overNa₂SO₄, filtered and concentrated in vacuo to afford5-(5-amino-2-Isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (2.156g, 82%) as a dark-orange solid.

Step 2.1-[5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-3-ethyl-urea

A solution of5-(5-amino-2-Isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (0.117g, 0.4 mmol) and ethyl isocyanate (0.034 g, 0.5 mmol) in 4 mL of toluenewas heated to 100° C. in a sealed tube. After 5 h the solution wascooled and concentrated in vacuo gave a brown solid. Purification viaflash chromatography (CH₂Cl₂/MeOH 97:3) afforded1-[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-3-ethyl-urea(0.120 g, 83%) as a white solid; (M+H)=361.

Example 121-[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-3-phenyl-urea

5-(5-amino-2-Isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (0.309g, 1.1 mmol) was converted, as described in the above procedure, to1-[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-3-phenyl-urea(0.122 g, 28%) as white solid; [MH]⁺=408.

Similarly prepared from5-(5-amino-2-Isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (0.313g, 1.1 mmol) and 2,5-hexanedione (0.14 ml, 1.2 mmol) was5-[5-(2,5-Dimethyl-pyrrol-1-yl)-2-isopropyl-4-methoxy-phenoxy]-pyrimidine-2,4-diamine,(0.259 g, 64%). (M+H)=368.

Example 134-Chloro-N-[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-butyramide

To a solution of5-(5-amino-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (0.400g, 1.4 mmol) in 15 ml CHCl₃ and Na₂HPO₄ (0.392 g, 2.8 mmol) was added4-chlorobutyryl chloride (0.194 g, 1.4 mmol) drop-wise. After 4.5 h, H₂Oand CH₂Cl₂ were added and the mixture was allowed to stir 15 min. Themixture was neutralized with 2N Na₂CO₃ and extracted with CH₂Cl₂. Thecombined organics were washed with brine, dried over Na₂SO₄, filteredand concentrated in vacuo to afford4-chloro-N-[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-butyramide(0.495 g, 91%) as brown foam; [MH]⁺=394.

Example 145-(2-Isopropyl-5-isothiocyanato-4-methoxy-phenoxy)-pyrimidine-2,4-diamine

To a solution of5-(5-amino-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine (0.100g, 0.4 mmol) in 1 ml H₂O and TFA (0.040 g, 0.4 mmol) was addedthiophosgene (0.040 g, 0.4 mmol). After 1 h the mixture was neutralizedwith 2M NaOH and extracted with CH₂Cl₂. The combined organics werewashed with brine, dried over Na₂SO₄, filtered and concentrated in vacuoto afford5-(2-isopropyl-5-isothiocyanato-4-methoxy-phenoxy)-pyrimidine-2,4-diamine(0.042 g, 36%) as brown foam [MH]⁺=334.

Example 152-[5-(2,4-Diaminopyrimidin-5-yloxy)-4-isopropyl-2methoxy-phenyl]-propan-2-ol

To a solution of methylmagnesium bromide (83.4 mmol, 27.8 ml, 3.0 M inEt₂O) in 83 mL THF at 0° C. was added1-[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-ethanone(2.523 g, 8.3 mmol, from Example 16) in portions. After 16 h the mixturewas cooled to 0° C. and was quenched by the addition 10% NH₄Cl. H₂O wasadded and the mixture was extracted with ethyl acetate. The combinedorganics were washed with H₂O, washed with brine, dried over NaHCO₃,filtered and concentrated in vacuo. The crude solid was taken up in 31ml DMF. K₂CO₃ (0.65 g, 4.7 mmol) and iodomethane (0.098 ml, 1.6 mmol)were added and the mixture was warmed to 50° C. Additional portions ofiodomethane (0.019 mL, 0.6 mmol) was added at 1, 2 and 3 hr. After 16 hthe mixture was cooled and 10% NH₄Cl and extracted with ethyl acetate.The combined organics were washed with H₂O, washed with brine, driedwith Na₂SO₄, filtered and concentrated in vacuo to give2-[5-(2,4-diaminopyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-propan-2-ol(0.711 g, yield) as a white solid. [MH]⁺=333.

Example 16 5-(2,5-Diiosopropyl-methoxy-phenoxy)-pyrimidine-2,4-diamine

To a solution of2-[5-(2,4-diaminopyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-propan-2-ol:(0.350 g, 1.1 mmol) in 10 ml CH₂Cl₂ was added trifluoroacetic acid (4.0ml, 52.6 mmol) and triethylsilane (1.7 ml, 10.5 mmol). After 30 minsaturated NaHCO₃ was added and the mixture was extracted with ethylacetate. The combined organics were washed with brine, dried overNa₂SO₄, filtered and concentrated in vacuo to give a crude oil.Purification via flash chromatography (96:4 CH₂Cl₂/MeOH) gave5-(2,5-diiosopropyl-methoxy-phenoxy)-pyrimidine-2,4-diamine (0.225 g,68%) as a white solid. [MH]⁺=317.

Example 171-[5-(2,4-Diamino-pyrimidine-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-ethanol

To a solution of1-[5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-ethanone(2.500 g, 8.3 mmol) in 100 ml MeOH was slowly added NaBH₄ (1.566 g, 41.4mmol) at 0° C. The solution was allowed to warm to rt. After 20 h, thesaturated NH₄Cl was added, the mixture was concentrated in vacuo andextracted with ethyl acetate. The combined organics were washed withbrine, dried over Na₂SO₄, filtered and concentrated in vacuo.Purification via silica gel column chromatography (9:1 CH₂Cl₂/MeOH)afforded to1-[5-(2,4-diamino-pyrimidine-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-ethanol(1.613 g, 60%) as white foam; [MH]⁺=301.

Example 185-(2-Isopropyl-4-methoxy-5-vinyl-phenoxy)-pyrimidine-2,4-diamine and5-[2-Isopropyl-4-methoxy-5-(1-methoxy-ethyl)-phenoxy]-pyrimidine-2,4-diamine

To a solution of1-[5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-phenyl]-ethanol(1.613 g, 5.3 mmol) in 30 ml CH₂Cl₂ at −78° C. was added DAST (0.935 g,5.8 mmol). After stirring 1.5 h, saturated NaHCO₃ was added and themixture was extracted by CH₂Cl₂. The combined organics were washed withbrine and dried Na₂SO₄, filtered and concentrated in vacuo. Purificationvia silica gel chromatography (95:5 CH₂Cl₂/MeOH) gave5-(2-Isopropyl-4-methoxy-5-vinyl-phenoxy)-pyrimidine-2,4-diamine (0.044g, 3%) as a foam ([MH]⁺=301) and5-[2-Isopropyl-4-methoxy-5-(1-methoxy-ethyl)-phenoxy]-pyrimidine-2,4-diamine(0.075 g, 4%) as foam. [MH]⁺=303.

Example 205-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-N-methyl-benzenemethylsulfonamideStep 1.5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzenesulfonylchloride

A mixture of pyrimidine (0.400 g, 1.5 mmol) in 2 ml chlorosulfonic acidwas allowed to stir 20 min. The mixture was poured over ice. Theprecipitate was filtered, washed by cold H₂O and dried under vacuum toafford5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzenesulfonylchloride (0.515 g, 95%) as a white solid; [MH]⁺=373.

Step 2.5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-N-methyl-benzenemethylsulfonamide

To 10 ml methyl amine −78° C. in a screw-capped tube was added5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzenesulfonylchloride (0.300 g, 0.8 mmol). The mixture was allowed to warm to roomtemperature. After 20 hours the mixture was evaporated, washed with H₂O,and dried under vacuum to afford5-(2,4-diamino-pyrimidin-5-yloxy)-4-idopropyl-2-methoxy-N-methyl-benzenemethylsulfonamide(0.170 g, 57%) as a white solid; mp (HCl salt)=252.3-252.9° C.;[MH]⁺=367.

Similarly prepared, replacing methylamine with ethylamine, was5-(2,4-Diamino-pyrimidin-5-yloxy)-N-ethyl-4-isopropyl-2-methoxy-benzenesulsonamide(0.186 g, 61%) as a white solid; mp (HCl salt)=260-265° C.; [MH]⁺=382.

Example 215-(2,4-Diamino-pyrimidin-5-yloxy)4-isopropyl-2-methoxy-N,N-dimethyl-benzamide

To a suspension of5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzoic acid(180 mg, 0.57 mmol, from Example 17) in anhydrous dichloromethane (5.6mL) was added trifluoroacetic acid (0.08 mL, 1.14 mmol) and then thionylchloride (0.36 mL, 5.65 mmol). After 1 hour the reaction wasconcentrated. To the residue was added anhydrous dichloromethane (4.5mL) and dimethylamine (2.84 mL of a 2M solution in tetrahydrofuran, 5.65mmol). After 2 hours stirring at room temperature, the reaction wasfiltered and concentrated. Purification via silica gel columnchromatography eluting with 95/5/0.1 to 93/7/0.1dichloromethane/methanol/ammonium hydroxide yielded5-(2,4-diamino-pyrimidin-5-yloxy)4-isopropyl-2-methoxy-N,N-dimethyl-benzamide(40 mg, 20%) as pale yellow solid, MS (M+H)=346.

Similarly prepared using methylamine instead of dimethylamine,5-(2,4-diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-N-methyl-benzamide(23 mg, 15%) was prepared as pale yellow solid, MS (M+H)=332.

Example 22 4-(2,4-Diamino-pyrimidin-5-yloxy)-2-iodo-5-isopropyl-phenol

To a cold suspension of 1 (0.21 g, 0.52 mmol) in dichloromethane (15 ml)at 0° C. was added BBr3 (0.26 g, 1.05 mmol). The reaction mixture wasstirred at room temperature for 16 hrs., quenched with water andbasified with sat. NaHCO3. The insoluble solid was collected byfiltration. The filtrate was washed with water, dried over Na₂SO₄,filtered and concentrated in vacuo. The combined residue was purifiedvia flash chromatographed on silica gel (3 to 5% methanol indichloromethane with 0.1% NH₄OH) gave desired product (0.174 g, 86%),(M+H)=387.

Example 235-(5-Iodo-2-isopropyl-4-prop-2-ynyloxy-phenoxy)-pyrimidine-2,4-diamine

To 4-(2,4-Diamino-pyrimidin-5-yloxy)-2-iodo-5-isopropyl-phenol (200 mg,0.43 mmol) dissolved in anhydrous N,N-dimethylformamide (2 mL) was addedanhydrous potassium carbonate (414 mg, 3.00 mmol) and propargyl chloride(0.03 mL, 0.43 mmol). After stirring at room temperature overnight, thereaction was extracted with dichloromethane, water and brine. Thedichloromethane layer was dried using anhydrous magnesium sulfate,concentrated, and purified via silica gel column chromatography(95/5/0.1 dichloromethane/methanol/ammonium hydroxide) to yield5-(5-iodo-2-isopropyl-4-prop-2-ynyloxy-phenoxy)-pyrimidine-2,4-diamineas white solid (131 mg, 71%), MS (M+H)=425.

Example 245-(5-Ethanesulfonyl-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine

To a solution of sodium sulfite (541 mg, 4.29 mmol) in water (20 mL) wasadded5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzenesulfonylchloride (400 mg, 1.07 mmol) and the reaction was heated at 80° C. for 1hour. Sodium bicarbonate (361 mg, 4.29 mmol-dissolved in 5 mL water),dioxane (20 mL), and ethyl iodide (0.10 mL, 1.29 mmol) were added andthe reaction was heated at 80° C. for 2 hours. The reaction wasconcentrated, extracted with dichloromethane (150 mL) and water (20 mL).The dichloromethane layer was dried using anhydrous sodium sulfate,concentrated, and purified via silica gel column chromatography(95/5/0.1 dichloromethane/methanol/ammonium hydroxide) to yield5-(5-ethanesulfonyl-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine(77 mg, 20%) as white solid, MS (M+H)=367.

Example 255-(2-Isopropyl-4-methoxy-5-trifluoromethyl-phenoxy)-pyrimidine-2,4-diamine

The synthetic procedure used in this Example is outlined in Scheme E.

Step 1. 1-Iodo-4-isopropyl-2-methoxy-5-(toluene-4-sulfonyl)-benzene

To a solution of 2-Isopropyl-4-methoxy-1-(toluene-4-sulfonyl)-benzene(10 g, 31.25 mmol) in HOAc (10 ml) was added a solution of ICI (9.6 g,59.26 mmol) in HOAc (10 ml) and H₂O (5 ml). The reaction mixture wasstirred at room temperature for 16 hrs and basified by saturated NaHCO₃solution. The aqueous solution was extracted into EtOAc which was washedwith water, brine, dried over Na₂SO₄, filtered and concentrated in vacuoto give 1-Iodo-4-isopropyl-2-methoxy-5-(toluene-4-sulfonyl)-benzene(12.35 g, 89%).

Step 2.1-Isopropyl-5-methoxy-2-(toluene-4-sulfonyl)-4-trifluoromethyl-benzene

To a hot mixture of1-Iodo-4-isopropyl-2-methoxy-5-(toluene-4-sulfonyl)-benzene (0.5 g, 1.12mmol), CuI, KF in anhydrous DMF (10 ml) at 120° C. oil bath temperature,was added trifluoromethyl iodide (0.64 g, 4.48 mmol) in portions over 30min. The reaction mixture was heated for 4 hrs and poured into H₂O (100ml). The insoluble solid, which was collected by filtration wastriturated with methylene chloride, filtered and concentrated to give1-Isopropyl-5-methoxy-2-(toluene-4-sulfonyl)-4-trifluoromethyl-benzene(0.45 g, 100%) as a solid.

Step 3. 2-Isopropyl-4-methoxy-5-trifluoromethyl-phenol

A solution of1-Isopropyl-5-methoxy-2-(toluene-4-sulfonyl)-4-trifluoromethyl-benzene(0.40 g, 1.03 mmol) and NaOH (0.5 g, 12.5 mmol) in MeOH (5 ml) and H₂O(5 ml) was heated at 90° C. for 2 hrs. The cooled reaction mixture wasacidified with 3N HCl and extracted into methylene chloride. Thecombined extracts was dried with Na₂SO₄, filtered and concentrated togive desired 2-Isopropyl-4-methoxy-5-trifluoromethyl-phenol (0.194 g,81%) as an oil.

Step 4.5-(2-Isopropyl-4-methoxy-5-trifluoromethyl-phenoxy)-pyrimidine-2,4-diamine

Following the procedure of Example 2 steps 5-7,2-Isopropyl-4-methoxy-5-trifluoromethyl-phenol was converted to5-(2-Isopropyl-4-methoxy-5-trifluoromethyl-phenoxy)-pyrimidine-2,4-diamine.(M+H)=343

Example 265-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-thiobenzamide

A mixture of5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-benzamide (0.25g, 0.79 mmol, prepared according to the procedure of Example 52) andLawesson's reagent (0.96 g, 2.37 mmol) in anhydrous THF (20 ml) wasstirred at room temperature for 16 hrs and concentrated in vacuo. Flashchromatography on silica (5% CH₃OH in methylene chloride with 1% NH₄OH)gave5-(2,4-Diamino-pyrimidin-5-yloxy)-4-isopropyl-2-methoxy-thiobenzamide(0.201 g, 76%) as a yellow solid.

Example 275-(4-Ethoxy-5-iodo-2-isopropyl-phenoxy)-pyrimidine-2,4-diamine

To a solution of4-(2,4-Diamino-pyrimidin-5-yloxy)-2-iodo-5-isopropyl-phenol (0.2 g, 0.52mmol) in anhydrous DMF (2 ml) was added EtBr (57 mg, 0.52 mmol) inportions. The reaction mixture was partitioned between EtOAc and H₂O.The organic extract was dried over Na₂SO₄, filtered and concentrated.Flash chromatography on silica (3% MeOH in methylene chloride with 1%NH₄OH) gave5-(4-Ethoxy-5-iodo-2-isopropyl-phenoxy)-pyrimidine-2,4-diamine (0.17 g,28%) as a yellow solid. (M+H)=415.

Example 28 Diaminopyrimidine Antagonists of P2X3 and P2X2/3 ATP-GatedChannels Produce an Effective Suppression of Systolic and DiastolicBlood Pressure in the Spontaneously Hypertensive Rats (SHRs)

The effect of aryloxydiaminopyrimidine P2X3 receptor antagonists (acompound of formula (I)) in the sensitization of these afferents in SHrats was investigated. Studies were performed to investigate theefficacy of intravenous administration of selective doses of theseantagonistic compounds in SH rats as a model for human disease lookingat instrumentally monitored blood pressure, sympathetic nerve discharge,and excitability of petrosal chemosensory carotid sinus afferents.

Evidence suggests that P2X3-containing receptors are expressed bypetrosal carotid sinus chemosensory afferents and contribute to theirhyperexcitability and hypertonicity. Initial findings illustrated inFIG. 1 (A-C) had indicated that inhibition of P2X3 receptors with thebenzyl-diaminopyrimidine antagonist Compound A led to attenuation oftonic firing of carotid sinus afferents and excitatory responses tohypoxia or ATP (and analogs); these effects were noted in wild-typemice, and were more sensitively observed in P2X2 knock-out (KO) micereflecting a role of homotrimeric P2X3 receptors in the P2X2-KO; in theP2X3 KO mouse, responses to hypoxia and ATP were no longer sensitive toinhibition by Compound A, confirming the critical involvement of P2X3subunit containing receptors in mediating these effects. Thus, theeffect of aryloxydiaminopyrimidine P2X3 receptor antagonists (Compound15; Compound 16) in the sensitization of these afferents in SH rats wasinvestigated. Studies were performed to investigate the efficacy ofintravenous administration of selective doses of these antagonisticcompounds in SH rats looking at instrumentally monitored blood pressure,sympathetic nerve discharge, and excitability of petrosal chemosensorycarotid sinus afferents.

Summary of investigations and key results.

In the SH rat, denervation of the carotid bodies bilaterally produced aprecipitous fall in arterial pressure (FIG. 2A); no such effect wasobserved in normotensive Wistar rat. Moreover, there was ˜55% reductionin sympathetic discharge following denervation of the carotid bodies.These data generated the hypothesis that in addition to the heightenedperipheral chemoreflex sympathoexcitatory response in SH versus Wistarrats (Tan et al. 2010, Circulation Research 106, 536-545), there wasalso aberrant discharge emanating from the carotid body of SH rats.

Intracellular recordings from petrosal ganglion chemoreceptive primaryafferents confirmed the presence of aberrant chemoreceptor tonicitypresent in SH rats (FIG. 2B; Moraes & Paton—unpublished data). Inaddition, SH rat chemosensitive petrosal neurones were more depolarizedand showed exaggerated responses to stimuli compared to those recordedfrom normotensive rats (FIG. 2B).

Administration of P2X3 antagonist (a compound of formula (I); 1 mg·kg⁻¹i.v.) reduced arterial pressure in SH rats but had no effect in Wistarnormotensive rats (FIG. 2C; Pijacka & Paton—unpublished data).Importantly, the reduction in arterial pressure in SH rats was dependenton intact chemoreceptors as bilateral carotid ablation abolished theeffect of the compound of formula I. These data from a relevant modelfor human disease support the notion that blockade of P2X3 receptorsprovides a novel form of anti-hypertensive therapy that acts by blockingcarotid body aberrant discharge, and in turn reduces excess sympatheticdischarge.

FIG. 2B shows that the aberrant discharge of chemosensitive petrosalneurons in SH rats can be completely blocked by a compound of formula(I), a P2X3 antagonist, yet these neurones maintain an ability torespond to a chemoreceptor stimulus. This is important as it effectivelypermits maintenance of the ability to sense hypoxia/hypercapnia and lowplasma pH but prevents the aberrant discharge causing sympathoexcitationand the pathological effects this causes.

It is known that mammalian glomus cells release ATP when exposed tohypoxia and other chemical signals. FIG. 2D depicts the presence of P2X3receptors in the carotid body taken from a hypertensive human subject.Note the pattern of expression is consistent with these receptors beingon primary afferent terminals.

Example 29 Exemplary Tablet Formulations

A compound of formula (I) (such as a compound of Table 1) or otherrepresentative diaminopyrimidine P2X3 antagonists may be suppliedformulated in a yellow, film-coated, oval-shaped tablet containing 10,30, 50, 75, 100 or 300 mg of compound. Tablets may be formulated withUSP/NF compendial grade lactose monohydrate, hydroxypropyl methylcellulose (HPMC or Hypromellose), croscarmellose sodium,microcrystalline cellulose (Avicel PH102), and magnesium stearate asdescribed in the Table 2 (below). Tablets are film-coated with OpadryYellow (Colorcon, Inc.) and packaged in HDPE bottles with childresistant caps and induction seals.

TABLE 2 Compound 16 (Compound of Formula (I): Quantitative TabletComposition (300 mg & Placebo) Amount for Amount for 300 mg PlaceboTablet Tablet Component Grade Function (mg) (mg) Intragranular Compound16 In house Active 300.0 0 (Formula (I)) (milled) Lactose USP/NF Diluent187.8 487.8 monohydrate Croscarmellose USP/NF Disintegrant 18.0 18.0sodium Hydroxypropyl USP/NF Binder 18.0 18.0 methyl celluloseExtragranular Croscarmellose USP/NF Disintegrant 12.0 12.0 sodiumMicrocrystalline USP/NF Diluent 60.0 60.0 Cellulose Magnesium USP/NFLubricant 4.2 4.2 Stearate Core Tablet 600 600 Film Coating OpadryYellow * Film-coat 18.0 18.0 03K12429 Sterile Water USP/NF GranulatingAs needed As needed for Irrigation Solution Total Weight 618 618 of FilmCoated Tablet * Opadry Yellow is composed of the following USP/NFexcipients: hypromellose, titanium dioxide, talc, triacetin and yellowiron oxide.

Example 30 P2X3/P2X2/3 FLIPR (Fluorometric Imaging Plate Reader) Assay

CHO-K1 cells were transfected with cloned rat P2X3 or human P2X2/3receptor subunits and passaged in flasks. 18-24 hours before the FLIPRexperiment, cells were released from their flasks, centrifuged, andre-suspended in nutrient medium at 2.5×10⁵ cells/ml. The cells werealiquoted into black-walled 96-well plates at a density of 50,000cells/well and incubated overnight in 5% CO₂ at 37° C. On the day of theexperiment, cells were washed in FLIPR buffer (calcium- andmagnesium-free Hank's balanced salt solution, 10 mM HEPES, 2 mM CaCl₂,2.5 mM probenecid; FB). Each well received 100 μl FB and 100 μl of thefluorescent dye Fluo-3 AM [2 μM final conc.]. After a 1 hour dye loadingincubation at 37° C., the cells were washed 4 times with FB, and a final75 μl/well FB was left in each well.

Test compounds (dissolved in DMSO at 10 mM and serially diluted with FB)or vehicle were added to each well (25 μl of a 4X solution) and allowedto equilibrate for 20 minutes at room temperature. The plates were thenplaced in the FLIPR and a baseline fluorescence measurement (excitationat 488 nm and emission at 510-570 nm) was obtained for 10 seconds beforea 100 μl/well antagonist or vehicle addition. The antagonist was a 2Xsolution of α,β-meATP producing a final concentration of 1 μM (P2X3) or5 μM (P2X2/3). Fluorescence was measured for an additional 2 minutes at1 second intervals after antagonist addition. A final addition ofionomycin (5 μM, final concentration) was made to each well of the FLIPRtest plate to establish cell viability and maximum fluorescence ofdye-bound cytosolic calcium. Peak fluorescence in response to theaddition of α,β-meATP (in the absence and presence of test compounds)was measured and inhibition curves generated using nonlinear regression.PPADS, a standard P2X antagonist, was used as a positive control.

Using the above procedure, compounds of the invention exhibited activityfor the P2X3 receptor. The compound4-(2,4-Diamino-pyrimidin-5-yloxy)-2-iodo-5-isopropyl-phenol, forexample, exhibited a pIC₅₀ of approximately 8.3 using the above assay.

EQUIVALENTS

While the present technologies, methods, and inventions have beendescribed with reference to the specific embodiments thereof, it shouldbe understood by those skilled in the art that various changes may bemade and equivalents may be substituted without departing from the truespirit and scope of the invention. In addition, many modifications maybe made to adapt a particular situation, material, composition ofmatter, process, process step or steps, to the objective spirit andscope of the present invention. All such modifications are intended tobe within the scope of the claims appended hereto.

What is claimed is:
 1. A method of treating hypertension in a subject,comprising identifying a subject diagnosed with hypertension andadministering to the subject a compound of formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomerthereof; wherein: R¹ is hydrogen or optionally substituted C₁-C₆ alkyl;and R² is C₁-C₆ alkyl; C₂-C₆ alkenyl; C₂-C₆ alkynyl; amino;aminosulfonyl; halo; amido; C₁-C₆ haloalkyl; C₁-C₆ alkoxy; C₁-C₆hydroxy; C₁-C₆ haloalkoxy; nitro; C₁-C₆ hydroxyalkyl; C₁-C₆ alkoxyalkyl;C₁-C₆ hydroxyalkoxy; C₂-C₆ alkynylalkoxy; C₁-C₆ alkylsulfonyl; C₆-C₁₀arylsulfonyl; C₁-C₆ carboxyalkyl; cyano or C₁-C₆ alkylcarbonyl.
 2. Themethod of claim 1 wherein said subject diagnosed with hypertension has asystolic blood pressure above 120 mmHg and/or a diastolic pressure aboveabout 80 mmHg.
 3. The method of claim 1 or 2, wherein the subjectdiagnosed with hypertension is diagnosed with chronic treatmentresistant hypertension.
 4. A method of treating heart failure in asubject, comprising identifying a subject diagnosed with heart failureand administering to the subject a compound of formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomerthereof; wherein: R¹ is hydrogen or optionally substituted C₁-C₆ alkyl;and R² is C₁-C₆ alkyl; C₂-C₆ alkenyl; C₂-C₆ alkynyl; amino;aminosulfonyl; halo; amido; C₁-C₆ haloalkyl; C₁-C₆ alkoxy; C₁-C₆hydroxy; C₁-C₆ haloalkoxy; nitro; C₁-C₆ hydroxyalkyl; C₁-C₆ alkoxyalkyl;C₁-C₆ hydroxyalkoxy; C₂-C₆ alkynylalkoxy; C₁-C₆ alkylsulfonyl; C₆-C₁₀arylsulfonyl; C₁-C₆ carboxyalkyl; cyano or C₁-C₆ alkylcarbonyl.
 5. Themethod of claim 4, wherein said subject diagnosed with heart failure isdiagnosed with systolic heart failure.
 6. The method of claim 4, whereinsaid subject diagnosed with heart failure is diagnosed with diastolicheart failure.
 7. The method of claim 4, wherein said subject diagnosedwith heart failure is diagnosed with chronic heart failure.
 8. Themethod of claim 4, wherein said subject diagnosed with heart failure isdiagnosed with acute heart failure.
 9. A method of treating dyspnea orsleep apnea in a subject, comprising identifying a subject diagnosedwith dyspnea or sleep apnea and administering to the subject a compoundof formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomerthereof; wherein: R¹ is hydrogen or optionally substituted C₁-C₆ alkyl;and R² is C₁-C₆ alkyl; C₂-C₆ alkenyl; C₂-C₆ alkynyl; amino;aminosulfonyl; halo; amido; C₁-C₆ haloalkyl; C₁-C₆ alkoxy; C₁-C₆hydroxy; C₁-C₆ haloalkoxy; nitro; C₁-C₆ hydroxyalkyl; C₁-C₆ alkoxyalkyl;C₁-C₆ hydroxyalkoxy; C₂-C₆ alkynylalkoxy; C₁-C₆ alkylsulfonyl; C₆-C₁₀arylsulfonyl; C₁-C₆ carboxyalkyl; cyano or C₁-C₆ alkylcarbonyl.
 10. Themethod of claim 9 for treating sleep apnea in a subject, comprisingidentifying a subject diagnosed with sleep apnea and administering tothe subject a compound of formula (I):

or a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomerthereof; wherein: R¹ is hydrogen or optionally substituted C₁-C₆ alkyl;and R² is C₁-C₆ alkyl; C₂-C₆ alkenyl; C₂-C₆ alkynyl; amino;aminosulfonyl; halo; amido; C₁-C₆ haloalkyl; C₁-C₆ alkoxy; C₁-C₆hydroxy; C₁-C₆ haloalkoxy; nitro; C₁-C₆ hydroxyalkyl; C₁-C₆ alkoxyalkyl;C₁-C₆ hydroxyalkoxy; C₂-C₆ alkynylalkoxy; C₁-C₆ alkylsulfonyl; C₆-C₁₀arylsulfonyl; C₁-C₆ carboxyalkyl; cyano or C₁-C₆ alkylcarbonyl.
 11. Themethod of claim 10, wherein said subject diagnosed with sleep apnea isdiagnosed with central sleep apnea.
 12. The method of claim 10, whereinsaid subject diagnosed with sleep apnea is diagnosed with obstructivesleep apnea.
 13. The method of claim 10, wherein said subject diagnosedwith sleep apnea is diagnosed with mixed sleep apnea.
 14. The method ofclaim 10, wherein said subject diagnosed with sleep apnea is diagnosedas having more than about 5 apneic events per hour of sleep.
 15. Themethod of claim 10, wherein said subject diagnosed with sleep apnea isdiagnosed as having more than about 15 apneic events per hour of sleep.16. The method of claim 10, wherein said subject diagnosed with sleepapnea is diagnosed with as having more than about 25 apneic events perhour of sleep.
 17. The method of claim 1, wherein the compound offormula (I) is selected from the group consisting of:


18. The method of claim 2, wherein the subject diagnosed withhypertension is diagnosed with chronic treatment resistant hypertension.19. The method of claim 1, wherein the compound of formula (I) isselected from the group consisting of:


20. The method of claim 9, wherein the compound of formula (I) isselected from the group consisting of: